Effect of cooling rate and cryoprotectant concentration on intracellular ice formation of small abalone (Haliotis diversicolor) eggs

Cryobiology 67 (2013) 7–16

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Effect of cooling rate and cryoprotectant concentration on intracellular ice formation of small abalone (Haliotis diversicolor) eggs Chiang-Yi Yang a,c,1, Yu-Hui Flora Yeh a,1, Po-Ting Lee c, Ta-Te Lin a,b,⇑ a

Department of Bio-Industrial Mechatronics Engineering, National Taiwan University, Taipei, Taiwan, ROC Graduate Institute of Brain and Mind Science, National Taiwan University, Taipei, Taiwan, ROC c Department of Bio-Mechatronics Engineering, National I-Lan University, I-Lan, Taiwan, ROC b

a r t i c l e

i n f o

Article history: Received 24 January 2013 Accepted 12 April 2013 Available online 22 April 2013 Keywords: Cryopreservation TEC cryomicroscope Intracellular ice formation Small abalone

a b s t r a c t The intracellular ice formation (IIF) behavior of Haliotis diversicolor (small abalone) eggs is investigated in this study, in relation to controlling the cooling rate and the concentration of dimethyl sulfoxide (DMSO). The IIF phenomena are monitored under a self-developed thermoelectric cooling (TEC) cryomicroscope system which can achieve accurate temperature control without the use of liquid nitrogen. The accuracy of the isothermal and ramp control is within ±0.5 °C. The IIF results indicate that the IIF of small abalone eggs is well suppressed at cooling rates of 1.5, 3, 7 and 12 °C/min with 2.0, 2.5, 3.0 and 4.0 M DMSO in sea water. As 2.0 M DMSO in sea water is the minimum concentration that has sufficient IIF suppression, it is selected as the suspension solution for the cryopreservation of small abalone eggs in order to consider the solution’s toxicity effect. Moreover, IIF characteristics of the cumulative probability of IIF temperature distribution are shown to be well fitted by the Weibull probabilistic distribution. According to our IIF results and the Weibull distribution parameters, we conclude that cooling at 1.5 °C/min from 20 to 50 °C with 2.0 M DMSO in sea water is more feasible than other combinations of cooling rates and DMSO concentrations in our experiments. Applying this protocol and observing the subsequent osmotic activity, 48.8% of small abalone eggs are osmotically active after thawing. In addition, the higher the cooling rate, the less chance of osmotically active eggs. A separate fertility test experiment, with a cryopreservation protocol of 1.5 °C/min cooling rate and 2.0 M DMSO in sea water, achieves a hatching rate of 23.7%. This study is the first to characterize the IIF behavior of small abalone eggs in regard to the cooling rate and the DMSO concentration. The Weibull probabilistic model fitting in this study is an approach that can be applied by other researchers for effective cryopreservation variability estimation and analysis. Ó 2013 Elsevier Inc. All rights reserved.

Introduction Haliotis diversicolor, or small abalone, is a shellfish found in the waters around Taiwan; it has a high economic value in both domestic and foreign markets [8]. However, devastating deaths of small abalone eggs for unidentifiable reasons have occurred since 2001 and, as a consequence, the small abalone industry has been badly affected. Cryopreservation is considered an effective strategy for avoiding sudden disease outbreaks. In Taiwan, small abalone sperm cryopreservation is well established [29], but only a few researchers have studied egg and embryo cryopreservation [4]. The storage of cryopreserved sperm and eggs would bring many benefits to the aquaculture industry, such as the reduction

⇑ Corresponding author. Address: Department of Bio-Industrial Mechatronics Engineering, National Taiwan University, No. 1, Sec. 4, Roosevelt Rd., Taipei 106, Taiwan, ROC. Fax: +886 2 23929416. E-mail address: [email protected] (T.-T. Lin). 1 These authors contributed equally to this work. 0011-2240/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cryobiol.2013.04.003

of brood stock conditioning costs and selective breeding; hence, there is considerable motivation for the effective cryopreservation of small abalone eggs. In our previous study, we reported on the fundamental experimental results of the osmometric characteristics of small abalone eggs [14]. This research work provided the basic information needed for further investigation in developing a feasible cryopreservation protocol for small abalone eggs. In a conventional cryopreservation protocol, the cooling rate and concentration of cryoprotectant (CPA) are two important factors that affect the survival rate after cryopreservation [16–18]. The mechanisms of freezing injury are conventionally considered a solution toxicity effect (STE) or an intracellular ice formation (IIF), based on the rate at which cells are frozen [1–3,10,33]. During slower freezing, extracellular ice crystallization creates a driving force to efflux intracellular water, which causes a high STE inside the cell or excessive shrinkage of the cell. CPAs can protect cells from STE; however, they can also kill cells at inappropriately high concentrations. In contrast, if the freezing rate is too fast, the cell does not have sufficient time to release the water, and it will be

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Fig. 1. Schematic setup of the TEC cryomicroscope system.

Fig. 2. Detailed schematic setup of the TEC cold stage.

supercooled and eventually frozen. This phenomenon, known as IIF, has proven to be lethal for cells. The IIF phenomenon can be observed under a cryomicroscope as a sudden flashing of cytoplasm during freezing [27]. IIF events can be recorded and analyzed to yield information that could facilitate the design of a successful cryopreservation protocol through adjustments to the cooling rate and the initial concentration of CPA. By using such an optimized cryopreservation procedure, the occurrence of IIF and the extent of STE would be minimized. Vitrification and slow freezing are practical methods for the cryopreservation of mammalian oocytes and embryos [7,9,12,15,22–24,26,30,33]. Vitrification is easy to implement with a low consumption of liquid nitrogen as it lowers the cooling temperature and increases viscosity. However, as permeating cryoprotectants are added to cells at a high concentration to protect against IIF, this creates substantial toxicity to the cell at room temperature; thus, only a very short time is allowed for equilibration before freezing at an extremely rapid cooling rate. For a slow freezing approach, IIF can be avoided by the increase of intracellular concentration, due to the efflux of water from cells during freezing when an optimum cooling rate is applied. The survival rate of cryopreserved cells greatly depends on the cooling rate used during freezing before the cells are plunged into liquid nitrogen. Therefore, when using the slow freezing approach, the observation and characterization of the IIF behaviors of small abalone eggs can facilitate the development of successful cryopreservation procedures. The optimum cooling rate can be systematically selected based on the observed IIF data and analyses.

In recent years, techniques, such as the thermoelectric cooling chip (TEC), have provided an accurate temperature control for cryomicroscope systems. In our previous works, a series of cryomicroscope systems was developed with TEC cold stages [5,31,32]. These TEC cryomicroscopes could carry out precise temperature control in simple operational procedures. This provided an opportunity to develop a small, easily set up operation and a cheap cryomicroscope system for studying the mechanisms of biological cells during cryopreservation procedures. The aims of this study were: (1) to investigate the effects of cooling rate and CPA concentration on the IIF phenomenon of small abalone eggs using the self-developed TEC cryomicroscope system; (2) to characterize the IIF behaviors of small abalone eggs with the experimental data recorded by the TEC cryomicroscope system; hence, the appropriate initial concentration of CPA and optimal freezing rate are determined; and (3) to examine the hatching rate of the fertility test under the cryopreservation protocol designed based on the determined IIF characteristics. Materials and methods TEC cryomicroscope A cryomicroscope system was constructed in this study composed of a TEC cold stage, a temperature-control module, a self-designed current reversible circuit, an image processing module and software developed in LabVIEW to integrate the cryomicroscope system hardware (Fig. 1). The temperature-control module had a GWPS320 programmable power supply (GW Instek, pst-3202, Taiwan) connected to a COM port of a laptop computer. The reverse current circuit was self-designed with two solenoid valve relays that connected to the GWPS320. The main circuit and the control circuit were connected to channel 1 and channel 2 of the GWPS320, respectively. The circuit switched the direction of the direct current to control the heating or the cooling of the TEC cold stage. Additionally, the image processing module was a CCD microscope (Ching Hsing Computer-Tech, Ltd., Finescope FS-180, Taiwan) connected to a USB port of a laptop computer. As shown in Fig. 2, a TEC chip was adhered on the cold stage as a heat sink and connected to the GWPS320. The temperature of the TEC cold stage was read by a thermal couple reader (National Instruments, NI 9162/9221, USA) and controlled by a laptop computer using the proportional–integral–derivative (PID) algorithm. The T-type thermal couple read the cold stage temperature, which was monitored and recorded. We also developed and applied a TEC water circulator with another TEC chip to dissipate heat generated

C.-Y. Yang et al. / Cryobiology 67 (2013) 7–16

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less. The spawning of both female and male abalones was induced in the light and temperature stimulation cycle; however, the reactions were quite different. The seawater turned brownish for female spawning, while milky for male spawning. One or two cycles of such light and temperature stimulation usually induced smooth spawning. Small abalone eggs were collected and all the IIF experiments were performed about 6 h after spawning took place. IIF characterization

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(B) Fig. 3. Temperature control performance of the TEC cold stage: (A) Constant cooling rate control, (B) Isothermal control.

from the TEC chip on the cold stage. The TEC employed the Peltier effect to create a heat flux in the junction of two different materials. With an electric current running through the chip, the heat was transferred from one side to the other side against the temperature gradient. In other words, the direct current direction was used to switch the hot and cold plates in the system. Also shown in Fig. 2, another two-stage TEC chip (30  30  9 mm) was adhered on the copper heat exchanger and connected to the TEC water circulator on its hot side. Hence, the heat from the TEC chip could be continuously transferred to the TEC water circulator and discharged into the air. Thus, a low temperature environment was created on the other side of TEC, allowing the freezing process of the small abalone eggs to be observed. In our IIF observation experiments, small abalone eggs were placed in a steel spacer of 600 lm thickness on the TEC chip and covered by a glass cover slip. At set times during the freezing and thawing processes, digital images of the small abalone eggs at set temperatures were acquired for the IIF characteristic analyses. Preparation of small abalone eggs Ripe small abalones were acquired from a private company in northeastern Taiwan and transported within 30 min in Styrofoam boxes to the laboratory. The female abalones were kept in a recirculating system of 250 L of filtered natural seawater. They were stimulated by changing the environmental light and temperature at midnight. The female and male abalones were stored in separated aquariums. They were firstly exposed under the sun for 1.5– 2.0 h in the empty aquarium (the seawater was drained out). Then they were placed in a dark room with filled seawater for 2.0 h, in the condition that the seawater was exchanged once after 1.5 h. The temperature of the seawater was increased to 24–25 °C and then restored back to 22 °C at a rate of 1 °C/h or

In this study, we focused on the effects of freezing rate and CPA concentration on the IIF phenomenon for small abalone eggs under our self-developed TEC cryomicroscope system. Dimethyl sulfoxide (DMSO) was selected as the CPA used in the experiments, as it is the most commonly used CPA for shellfish eggs or embryos [6,20,21]. A series of DMSO concentrations was predetermined with a 1.0 M DMSO increment interval. The DMSO was added to the seawater at given concentrations ranging from 1.0 to 4.0 M. Cooling processes were designed in 7 different freezing rates: 45, 25, 15, 12, 7, 3 and 1.5 °C/min. After being equilibrated with the predetermined DMSO concentration for 20 min at around 20 °C, small abalone eggs were cooled from 20 to 50 °C at the predetermined cooling rate and held at 50 °C for 10 min. We assumed that the eggs would survive subsequent rapid freezing to 196 °C in liquid nitrogen if they were slowly cooled to 50 °C without IIF. We did not test the survival of small abalone eggs stored in liquid nitrogen, as the difference of cooling to 50 °C or 55 °C then further down to 196 °C has proven to be negligible [13]. During freezing, extracellular ice nucleation was not intentionally induced. In the experiments, digital images were taken and the temperatures recorded when the IIF phenomenon occurred. About 10 eggs were frozen in an IIF experimental run, and at least 5 runs were carried out for each freezing rate. IIF phenomena were observed and the cumulative IIF probabilities were calculated. When a smaller interval of DMSO concentration increment was needed for better analysis, the concentration of DMSO was adjusted to increase at a 0.5 M DMSO interval instead of the initial 1.0 M. Osmotic activity observation From all cumulative probability plots of the IIF temperature distribution, the cooling rates that resulted in lower total cumulative probability of IIF were selected for use in further freezing experiments so the osmotic activity after thawing could be observed. The osmotic activity is defined as the eggs are capable of exhibiting osmotic behavior and restoring their shape back to the shape before freezing. The osmotic activity of small abalone eggs was observed by the TEC cryomicroscope at 500  magnification. The two lowest freezing rates were selected from the plots. Small abalone eggs were cooled from 20 to 50 °C at the selected freezing rates in the presence of the selected DMSO concentrations. When the temperature reached 50 °C, an isothermal process was held at 50 °C for 10 min. After that, a warming process was carried out at 112 °C/min to 25 °C and was held for another 10 min. The protocol which gained the highest osmotic activity rate was selected for the fertility test. Fertility test To compare the effects of the loading of cryoprotectant and the freezing on the fertility of small abalone eggs, eggs from 5 females were distributed equally in three containers with 150 cc of seawater in each container. Eggs in these three containers were comprised of fresh eggs, eggs after loading and unloading of 2 M

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(A)

(B)

(C)

(D)

(E)

(F)

Fig. 4. IIF images of small abalone eggs during cooling process under the TEC cryomicroscope: (A) Small abalone eggs before freezing, (B) 25% IIF occurred (5/20), (C) 50% IIF occurred (10/20), (D) 70% IIF occurred (14/20), (E) 85% IIF occurred (17/20), (F) 100% IIF occurred during freezing.

DMSO and eggs subjected to freezing to 50 °C, respectively. The eggs were fertilized with fresh sperm from three males, separately. The fertilization was then carried out at the ratio of ten spermatozoa to one egg in a beaker filled with seawater. After 15 h, eggs from each of the three containers were sampled ten times randomly into Petri dishes. The samples were observed under the microscope to determine the hatching rate, with the hatching rate here defined as the number of eggs that reached the trochophore larvae stage divided by the total number of eggs observed.

Results and discussion

perature system was performed with a mercury thermometer at the temperatures of 20 °C and 0 °C. Secondly, the PID control algorithm was employed in the system to ensure the temperature control accuracy with the designed freezing rate and in the isothermal process. Figs. 3(A) and (B) show the results of the temperature control performance test of our system at the designed freezing rate and in the isothermal process, respectively. Fig. 3(A) shows a number of lines representing cooling from 20 to 50 °C at different freezing rates. The differences of the real measured temperatures and the desired control temperature were all within the error range of ±0.5 °C. The operational temperature was controlled in the range of 70 to 55 °C for a freezing rate set between 1 and 80 °C/min. For the thawing process, the fastest thawing rate was set at 112 °C/min.

Temperature control of the TEC cryomicroscope IIF phenomenon observation Before IIF experiments can be performed, two key procedures must be executed. Firstly, the temperature reading from the T-type thermal couple should be calibrated. A linear calibration of our tem-

The formation of intracellular ice in a super-cooled biological cell causes light to scatter from the cytoplasm of the cell; thus,

C.-Y. Yang et al. / Cryobiology 67 (2013) 7–16

Cumulative Probability of IIF

1.0

11

50% at the same cooling rates as with 2.0 M DMSO; there was no IIF at a cooling rate of 1.5 °C/min (Fig. 6(D)). When the eggs were suspended in 3.0 M DMSO and cooled at 15 °C/min or slower, the cumulative IIF probabilities were all below 50%. At the cooling rates of 3 °C/min and 1.5 °C/min, no IIF was observed (Fig. 6(E)). Fig. 6(F) shows a similar result for the 4.0 M DMSO experiment. Additionally, no IIF was observed at the cooling rates of 7, 3 and 1.5 °C/min. From the cumulative probability plots, we realized that the DMSO concentration should be higher than 2.0 M in order to avoid IIF. Hence, the suspension solution was set as 2.0 M DMSO for the osmotic activity experiments.

0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 0

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Temperature (°C) Fig. 5. Cumulative probability of IIF temperature distributions for small abalone eggs in Taiwan. Samples were suspended in filtered sea water (without cryoprotectant) and cooled at 45( ), 25( ), 15( ), 12( ), 7( ), 3( ) and 1.5( ) °C/min cooling rates, respectively.

the refraction of the intracellular content shows significant changes under a cryomicro scope. In other words, the cell scatters light under high illumination when IIF occurs. The background (TEC surface in the cold stage) of the cell is painted black in advance using oil-based marker pen ink, so the scattered light can be observed like a flashing light bulb under the cryomicroscope. This phenomenon is called ‘‘flashing’’ and is an indicator of IIF [27]. Fig. 4 shows a typical example of IIF phenomenon observations of small abalone eggs going through different stages of the freezing process under the TEC cryomicroscope at 125  magnification. In each figure, the flashing light bulbs represent the eggs during the IIF phenomenon. Fig. 4(A) is an image of small abalone eggs before the freezing process; Figs. 4(B)–(F) show 25, 50, 70, 85 and 100% of small abalone eggs having IIF development, respectively. In our study, all experimental cumulative probabilities of IIF temperature distributions were calculated after the experiments. Figs. 5 and 6 illustrate the cumulative probabilities of IIF temperature distributions for small abalone eggs in the absence and in the presence of DMSO at different cooling rates, respectively. As shown in Fig. 5 (without DMSO), all total cumulative probabilities of IIF reached 100% when temperatures were far higher than 40 °C. Moreover, all these cumulative probability curves had a similar pattern, which indicated that small abalone eggs could not be preserved without CPA. Furthermore, without CPA, adjusting the cooling rate had no obvious effect on suppressing the IIF incidence. The slope of a cumulative IIF probability curve quantifies the degree of the IIF being suppressed. A declining slope means that the suppression of IIF occurrence is promoted. As shown in Figs. 5, 6(A) and 6(B), when the concentration of DMSO was raised from 0 to 1.5 M in the experiment, there were insignificant changes in the IIF suppression. Only slight decreases of the curve slope can be noted in the figures. All cumulative IIF probabilities still reached 100% before 40 °C in the experiments. This informed us that suppressing IIF by controlling the freezing rate was not effective when the DMSO concentration was in the range of 0–1.5 M. With 2.0 M DMSO, the cumulative probability curves of the 7 predetermined cooling rates started to show differences in the IIF temperature distribution, as shown in Fig. 6(C), indicating that the cooling rate began to have an effect on IIF occurrence when small abalone eggs were suspended in 2.0 M DMSO. We discovered that the total cumulative probabilities of IIF occurrence started to drop below 50% at cooling rates of 12, 7, 3 and 1.5 °C/min, as shown in Fig. 6(C). Similar IIF temperature distribution patterns were observed for DMSO concentrations higher than 2.0 M (Fig. 6(D)–(F)). With 2.5 M DMSO, the probability dropped below

Cumulative probability analysis of IIF temperature distribution The initial temperature of IIF occurrence provided a means to select the seeding temperature at a certain cooling rate. Fig. 7 shows the initial temperatures of IIF occurrence at different concentrations of DMSO. The seeding temperature was close to but higher than the initial temperature of IIF occurrence. IIF is lethal to biological cells and so served as an indicator for finding a feasible cryopreservation protocol. A feasible cryopreservation protocol with good suppression of IIF occurrence can be determined through cumulative IIF probability curves. When the cumulative probability curves of IIF temperature distribution were well separated in the plot, the IIF temperature distribution was cooling-rate dependent with the designed concentration of DMSO. In other words, the cooling rate had a significant effect on IIF occurrence. The median IIF temperature (TIIF50) is defined as the temperature at which the cumulative probability of IIF occurrence is 50% in a given experimental group. The median IIF temperature of each curve was calculated and summarized, as shown in Table 1. ND (not determined) denotes the case where the TIIF50 could not be determined as the IIF occurrence was below 50%. The possible range of a successful cryopreservation protocol could be determined from the summarized table if the suppression of IIF occurrence was the only considered criterion. Thus, from Table 1, the feasible cryopreservation protocol range was limited to cooling rates ranging from 1.5 to 12 °C/min with DMSO concentrations of 2.0–4.0 M. Furthermore, other achievable IIF suppression conditions were cooling at 15 °C/min with DMSO concentrations of 3.0 or 4.0 M. Weibull probabilistic model fitting Employing the Weibull distribution to carry out a probabilistic analysis of IIF occurrence temperature distribution was first proposed by Pitt and Steponkus [25]. The Weibull probabilistic model, which is a phenomenological model, can be used to fit the IIF likelihood during the freezing process. Although Karlsson’s model [11], which was extended from Toner’s model, is more widely used in simulations and in explaining freezing process mechanisms, the Weibull probabilistic model has the advantage of using fewer parameters for simulation. With the Weibull probabilistic model, the mean IIF temperature and the IIF behavior variation of a given population can be quantitatively analyzed. The model to describe the IIF characteristics when considering only the cooling process, as in our experiment, can be rewritten as:

1  P ¼ exp½ðT=T 0 Þr 

ð1Þ

where P is the cumulative probability of IIF upon cooling to temperature T °C, T is the temperature below the freezing point of water and T0 is the model parameter to be determined. Taking the natural

C.-Y. Yang et al. / Cryobiology 67 (2013) 7–16

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Fig. 6. Cumulative probability of IIF temperature distributions for small abalone eggs. Samples were suspended in (A) 1.0 M, (B) 1.5 M, (C) 2.0 M, (D) 2.5 M, (E) 3.0 M and (F) 4.0 M DMSO at 45( ), 25( ), 15( ), 12( ), 7( ), 3( ) and 1.5( ) °C/min cooling rates, respectively.

log and transpose on each side of Eq. (1), the Weibull model is transformed into a linear regression problem as in Eq. (2):

ln½lnð1  PÞ ¼ rlnT  rlnT 0

ð2Þ

In order to analyze the IIF behavior quantitatively, the cumulative probabilities of IIF temperature distribution were plotted on a Weibull scale, as shown in Fig. 8. The parameters T0 and r were estimated by linear regression on the cumulative probabilities of IIF temperature distribution using Eq. (2). In the Weibull probabilistic model, the scale parameter T0 is a location parameter closely related to the mean IIF temperature. The shape parameter r is in-

versely related to the coefficient of IIF temperature variation [25]. The larger value of T0 reflects a stronger depression in IIF temperature, and the larger value of r represents a smaller variation within the population. The linear regression results of the T0, r and R2 values obtained from the Weibull plots are summarized in Table 2. In our study, the cumulative probability of IIF temperature distribution for the 4.0 M DMSO experiments showed the best fit on the Weibull distribution. Cooling rates slower than 15 °C/min in the presence of 2.5 and 3.0 M DMSO, and cooling rates at 15 °C/ min and 25 °C/min in the presence of 0, 1.0, 1.5 and 2.0 M DMSO

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showed worse fits in the Weibull distribution. In general, the cumulative probability of IIF temperature distributions for small abalone eggs could be characterized by Weibull distributions, especially cooling rates between 1.5 and 12 °C/min with a DMSO concentration between 0 and 2.0 M. In addition, T0 values confirmed that the effect caused by the DMSO concentration variation was cooling-rate dependent. Besides, T0 values of the 2.0 M DMSO experiments were larger than the other concentrations for cooling rates slower than 12 °C/min. This meant that a 2.0 M DMSO concentration had a better effect on reducing the IIF temperature than other concentrations when the cooling rate was slower than 12 °C/min. For the shape parameter r, as shown in Table 2, the estimated values of r were consistent within each cooling rate across different DMSO concentrations. It was clear that with the same DMSO concentration, a higher cooling rate resulted in a larger r value; hence, less variability. However, at a 12 °C/min cooling rate with 2.5 M DMSO, the Weibull parameter r was not as large as the other parameters. This indicated that there was only a small variation in IIF temperature in our experimental data. For cooling at 1.5 °C/ min, the DMSO concentration had a significant effect on the mean IIF temperature. Thus, according to our experimental data, 1.5 °C/ min would be the optimal cooling rate for cryopreserving small abalone eggs. IIF behavior affected by cooling rates and DMSO concentrations To further investigate the composite effects of cooling rates and DMSO concentrations on the IIF behavior of small abalone eggs, we plotted the experimental data in Figs. 5 and 6 into a 3-dimensional plot of cumulative probability of IIF occurrence, as shown in Fig. 9. In Fig. 9(A), each node on the surface represents the experimental data on total cumulative probability undergoing a freezing process of 20 °C to 50 °C at a designed cooling rate with the designed DMSO concentration. The total cumulative probability of IIF (PTCIIF)

Fig. 8. Weibull plot of the inferred probability of instantaneous IIF versus transformed IIF for small abalone eggs in sea water at 45( ), 15( ), 12( ), 7( ), 3( ) and 1.5( ) °C/min cooling rates.

was 1.0 at cooling rates between 25 and 45 °C/min with 3.0 M DMSO or lower concentrations. The same results (PTCIIF = 1) were shown for all cooling rates with 1.5 M DMSO or lower concentrations. Hence, these cooling rates and DMSO settings were not appropriate cryopreservation protocols for small abalone eggs. As shown in Fig. 9(A), the slower the cooling rate and the higher concentration of DMSO, the better the suppression of IIF occurrence. The total cumulative probabilities of IIF occurrence with respect to different cooling rates and DMSO concentrations were estimated by the nearest neighbor grading method and plotted into a contour plot, as shown in Fig. 9(B). The region on the plot showing a total cumulative probability of IIF occurrence higher than 0.95 indicated that IIF occurrence could not be suppressed by adjusting the cooling rate or DMSO concentration. In other words, this region was concentration independent and also cooling-rate independent. As shown in Fig. 9(B), regions B and F were concentration dependent but cooling-rate independent. Hence, within these regions, increasing the DMSO concentration could suppress IIF occurrence, whereas decreasing the cooling rate had no obvious effect; in contrast, regions C and D were concentration independent but coolingrate dependent. The total cumulative probability of IIF occurrence was lower than 0.5 in region A. Therefore, the settings for a successful cryopreservation protocol for small abalone eggs could be delivered within the range of region A. To suppress IIF occurrence and avoid STE, we considered region E. At the left-hand side of region E, the lines of contour were very close to each other. However, the gaps between the lines of contour became larger when the DMSO concentration increased. In order to avoid STE, a low DMSO concentration was needed. That is to say, in region E, with only a small increase in DMSO concentration, the IIF occurrence could be suppressed more effectively. Hence, the settings in region E were selected for our further examination of the

Table 1 Observed median IIF temperature (TIIF50, °C) for small abalone eggs in seven designed concentrations of DMSO at seven designed cooling rates. Cooling rate

1.5 °C/min 3 °C/min 7 °C/min 12 °C/min 15 °C/min 25 °C/min 45 °C/min

Concentration Sea water

1.0 M

1.5 M

2.0 M

2.5 M

3.0 M

4.0 M

14.95 13.55 11.58 10.32 10.11 8.32 9.81

21.19 20.56 16.72 13.55 14.57 11.68 12.63

23.29 23.17 19.96 15.37 13.25 12.21 12.46

ND ND ND ND 24.16 14.95 14.81

ND ND ND ND 19.47 15.43 15.20

ND ND ND ND ND 20.54 20.21

ND ND ND ND ND 28.78 31.11

ND indicates TIIF50 could not be determined because incidence of IIF was 550%.

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Table 2 Weibull distribution test parameters for small abalone eggs cooled at 1, 3, 7, 12, 15, 25 and 40 °C/min and suspended in 0.0, 1.0, 1.5, 2.0, 2.5, 3.0 and 4.0 M DMSO, respectively. T0 (°C) r R2

Cooling rate (°C/min) 1.5

3

7

12

15

25

45

Concentration 0.0 M

16.97 4.09 0.927 22.91 4.94 0.951 25.60 4.45 0.980 49.58 4.67 0.947 –

15.04 3.84 0.949 21.93 5.23 0.989 24.71 5.98 0.978 53.26 3.63 0.925 51.92 2.78 0.740 –

12.05 7.10 0.969 18.75 4.36 0.984 22.87 4.31 0.972 49.14 3.51 0.900 31.69 3.44 0.894 –

10.52 5.74 0.921 15.13 3.92 0.920 17.81 3.75 0.941 45.96 2.38 0.944 28.46 1.97 0.270 40.71 2.76 0.463 38.72 12.11 0.940

11.20 7.96 0.864 16.67 5.68 0.813 14.93 6.20 0.882 28.49 3.49 0.788 24.12 3.44 0.421 31.07 4.40 0.465 38.72 10.85 0.917

9.24 7.17 0.893 12.08 15.45 0.917 13.49 6.26 0.782 17.01 7.27 0.898 15.82 14.89 0.906 20.98 14.75 0.910 29.42 12.29 0.931

10.07 16.59 0.918 13.38 9.30 0.779 13.10 12.63 0.918 15.59 14.28 0.732 15.48 25.84 0.880 21.95 11.74 0.876 31.50 7.62 0.904

1.0 M

1.5 M

2.0 M

2.5 M

3.0 M

–

4.0 M

–

–

–

cryopreservation of small abalone eggs. A 2.0 M DMSO concentration and 3 °C/min and 1.5 °C/min cooling rates were used as our cryopreservation protocols in order to study the viability of small abalone eggs after freezing to 50 °C/min by assessing their osmotic activity. A cooling rate of 1.5 °C/min with 1.5 M DMSO was selected as an improper protocol for purposes of comparison. Osmotic activity after cooling and thawing processes Three freezing protocols were examined for osmotic activity observations. In these three protocols, the temperature was cooled from 20 to 50 °C with different cooling rates and DMSO concentrations: 1.5 °C/min with 1.5 M DMSO, 1.5 °C/min with 2.0 M DMSO and 3 °C/min with 2.0 M DMSO. When the temperature reached 50 °C, all cooling processes were followed by a 50 °C isothermal process for 10 min. After the isothermal process, a rapid thawing at 112 °C/min was performed from 50 to 25 °C, and the temperature was then held at 25 °C for 10 min. The eggs were then ready for the osmotic activity observation at 500  magnification under the TEC cryomicroscope. As shown in Table 3, with cooling at 1.5 °C/min and 1.5 M DMSO, all abalone eggs were deformed and ruptured. This result was inconsistent with our expectation from the previous IIF observations, as shown in Fig. 6. In other words, 1.5 M DMSO was not an appropriate DMSO concentration as all eggs ruptured due to IIF during this freeze–thaw procedure. With freezing at 1.5 °C/min with 2.0 M DMSO, 48.8% of small abalone eggs still showed osmotic activity and could be restored back to their shape after thawing; however, only 30.9% survived freezing at 3 °C/min in the presence of 2.0 M DMSO. The result indicated that with 2.0 M DMSO, although the total cumulative IIF probabilities at 1.5 °C/min and 3 °C/min were close, a faster cooling speed had a worse effect on the osmotic activity of small abalone eggs. Thus, in order to set the base conditions for the subsequent fertility test, a 1.5 °C/min constant cooling rate and a 2.0 M DMSO concentration were selected. Fertility test For fertility tests, small abalone eggs were first equilibrated in 2.0 M DMSO for 20 min [14], then samples were placed in 1.5 ml microcentrifuge tubes to be frozen in a programmable refrigerated bath circulator. As mentioned above, a 1.5 °C/min constant cooling

rate and a 2.0 M DMSO concentration were set as the cryopreservation conditions. The samples were frozen to 50 °C and thawed in a water bath at room temperature. The thawed small abalone eggs were fertilized with fresh sperm and assessed as described previously. The results are shown in Table 4. The hatching rate of untreated fresh eggs was 71.3%, while there was a slight decrease of the hatching rate to 67.7% for eggs subjected to loading/unloading of 2 M DMSO. 16.1% of eggs subjected to CPA loading/unloading and freezing to 50 °C developed to the trochophore larvae stage after artificial fertilization. In comparison to the hatching rate of fresh eggs, a normalized hatching rate of 23.7% was achieved for small abalone eggs frozen to 50 °C in this study. Cryoinjury of small abalone eggs According to the IIF occurrence and osmotic activity observations shown in Table 3, under the cryopreservation protocol of a 3 °C/min cooling rate with 2.0 M DMSO, 21.4% of eggs had IIF and 69.1% of the non-IIF eggs did not show osmotic activity after thawing. Under another protocol of a cooling rate of 1.5 °C/min, IIF occurred in 17.1% of eggs and only 51.2% of the non-IIF eggs showed no osmotic activity after thawing. In other words, at least 47.7% and 34.1% of injured eggs were caused by other reasons when taking the non-IIF and osmotic active eggs out of consideration. We inferred that the osmotic pressure gradient (shrinkage and swelling of the membrane) played a role in this [19]. Although DMSO is considered to be an effective CPA to suppress IIF occurrence for shellfish gametes, recent papers have reported on other additives. For example, rare sugars can protect plasma membrane in the freezing and thawing processes and, hence, improve the survival rate [28]. However, further studies are necessary to evaluate the effectiveness of these methods and to investigate possible mechanisms of non-IIF injury. This research investigated the effects of the cooling rate and the DMSO concentration on the IIF behavior of small abalone eggs. Our experimental results have provided valuable details when determining the effective cooling rate for suppressing IIF occurrence and the initial DMSO concentration for avoiding STE in cryopreservation. Besides, osmotic activity after thawing could be observed by the self-developed TEC cryomicroscope system. These results have provided useful information for establishing a successful cryopreservation protocol for small abalone eggs. In addition, this

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C.-Y. Yang et al. / Cryobiology 67 (2013) 7–16

Cumulative IIF probability

1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2

0.0

0.1 0.0 45 3.0

25

15 12

Cooling rate (°C/min)

7

2.5

2.0

1.5

1.0

Concentration of DMSO (M)

3 1.5 4.0

(A)

(B) Fig. 9. Effect of cryoprotectant concentrations and cooling rates on small abalone eggs IIF: (A) 3D plot of the effect of interactions between cooling rate and concentration of DMSO on cumulative probability of IIF; and (B) contour plot of effect of cooling rates and DMSO concentrations on cumulative probability of IIF estimated by using nearest neighbor grading method.

Table 3 Observations of cumulative probability of IIF undergoing the cooling process and osmotic activity for Taiwan small abalone eggs after thawing processes. Selected cryopreservation protocol (Cooling rate/concentration of DMSO)

Cumulative probability of IIF

Rate of osmotic activity (number of eggs/number of eggs)

1.5 °C/min/1.5 M DMSO 3 °C/min/2 M DMSO 1.5 °C/min/2 M DMSO

1 0.214 0.171

0 (0/65) 0.309 (17/55) 0.488 (39/80)

Table 4 Comparisons of hatching rates of fresh eggs, eggs after loading and unloading of 2 M DMSO and eggs subjected to freezing to 50 °C at cooling rate of 1.5 °C/min.

Hatching rate Normalized hatching rate

Fresh eggs (number of eggs/number of eggs)

Eggs after loading/unloading of 2 M DMSO (number of eggs/number of eggs)

Eggs subjected to freezing to 50 °C (number of eggs/number of eggs)

71.3% (134/188) 100%

67.7% (130/192) 95%

16.1% (31/193) 23.7%

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research has helped accumulate basic information concerning the cryopreservation of gametes and embryos.

Conclusion In this study, we have not only characterized and parameterized the IIF behavior of small abalone eggs, but also developed a practical TEC cryomicroscope system. Our cryomicroscope system achieved precise temperature control without the use of liquid nitrogen. Also, the system featured modularized components and so was compact in size. The TEC cryomicroscope made it possible for the IIF phenomena and osmotic activity during the cryopreservation processes to be observed, recorded and analyzed. We have shown the effect of DMSO concentration on the IIF behavior of small abalone eggs. There was no dependence of the IIF temperature on the cooling rate over a DMSO concentration range of 0 M and 1.0 M. IIF was well suppressed at cooling rates ranging between 1.5 and 12 °C/min with a DMSO concentration between 2.0 and 4.0 M. According to our results, a 1.5 M DMSO or lower concentration in seawater was not suitable as the suspension medium for the cryopreservation of small abalone eggs. IIF occurrence was suppressed noticeably when the DMSO concentration was higher than 1.5 M. Based on the total cumulative probability contour plot of IIF associated with changes in cooling rate and DMSO concentration, the initial DMSO concentration for slow freezing could be determined. The protocol of a 1.5 °C/min constant cooling rate with 2.0 M DMSO was selected as the cryopreservation protocol for assessing the viability of small abalone eggs subjected to freezing. For osmotic activity observation, with freezing at 1.5 °C/min and 2.0 M DMSO, 48.8% of small abalone eggs were osmotically active and their shape was restored after thawing; however, in the case of freezing at 3 °C/min with 2.0 M DMSO, only 30.9% survived. The results indicated that faster freezing rates prompted fewer osmotically active eggs. In a separate fertility test experiment, with a 1.5 °C/min cooling rate and 2.0 M DMSO, a normalized hatching rate of 23.7% was achieved for small abalone eggs frozen to 50 °C. Furthermore, we applied the Weibull probabilistic analysis on IIF occurrence, which showed that the cumulative probability of IIF temperature distributions could be modeled with Weibull distributions. In particular, the Weibull distribution fit well in the region of cooling rates from 1.5 to 12 °C/min with a DMSO concentration of 0 M to 2.0 M. This study has provided detailed empirical results on the cryopreservation of small abalone eggs, including parameterized IIF observation. Different cryopreservation protocols for small abalone eggs were examined and the feasible ones have been identified, which is highly valuable information. With the self-developed TEC cryomicroscope system, a practicable cryopreservation system for small abalone eggs was built and put into practice. These results should be valuable guidelines for future researchers and industrial use. References [1] J.P. Acker, J. Elliott, L.E. McGann, Intercellular ice propagation: experimental evidence for ice growth through membrane pores, Biophys. J. 81 (2001) 1389– 1397.

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