A theoretically estimated optimal cooling rate for the cryopreservation of sperm cells from a live-bearing fish, the green swordtail Xiphophorus helleri

Abstract

Sperm cryopreservation of live-bearing fishes, such as those of the genus Xiphophorus is only beginning to be studied, although these fishes are valuable models for biomedical research and are commercially raised as ornamental fish for use in aquariums. To explore optimization of techniques for sperm cryopreservation of these fishes, this study measured the volumetric shrinkage response during freezing of sperm cells of Xiphophorus helleri by use of a shape-independent differential scanning calorimeter (DSC) technique. Volumetric shrinkage during freezing of X. helleri sperm cell suspensions was obtained in the presence of extracellular ice at a cooling rate of 20 degrees C/min in three different media: (1) Hanks' balanced salt solution (HBSS) without cryoprotective agents (CPAs); (2) HBSS with 14% (v/v) glycerol; and (3) HBSS with 10% (v/v) dimethyl sulfoxide (DMSO). The sperm cell was modeled as a cylinder of 33.3 microm in length and 0.59 microm in diameter with an osmotically inactive cell volume (V(b)) of 0.6V(o), where V(o) is the isotonic or initial cell volume. By fitting a model of water transport to the experimentally determined volumetric shrinkage data, the best-fit membrane permeability parameters (reference membrane permeability to water, L(pg) or L(pg)[cpa] and the activation energy, E(Lp) or E(Lp)[cpa]) of the Xiphophorus helleri sperm cell membrane were determined. The best-fit membrane permeability parameters at 20 degrees C/min in the absence of CPAs were: L(pg)=0.776 x 10(-15)m3/Ns (0.0046 microm/min atm), and E(Lp)=50.1 kJ/mol (11.97 kcal/mol) (R2=0.997). The corresponding parameters in the presence of 14% glycerol were L(pg)[cpa]=1.063 x 10(-15)m3/Ns (0.0063 microm/min atm), and E(Lp)[cpa]=83.81 kJ/mol (20.04 kcal/mol) (R2=0.997). The parameters in the presence of 10% DMSO were L(pg)[cpa]=1.4 x 10(-15)m3/Ns (0.0083 microm/min atm), and E(Lp)[cpa]=90.96 kJ/mol (21.75 kcal/mol) (R2=0.996). Parameters obtained in this study suggested that the optimal rate of cooling for X. helleri sperm cells in the presence of CPAs ranged from 20 to 35 degrees C/min and were in close agreement with recently published, empirically determined optimal cooling rates.

Fig. 1

Superimposed heat flow thermograms obtained during the initial (osmotically active cells; Curve A) and final (osmotically inactive cells; Curve B) cooling trials of X. helleri sperm cells at 20 °C/min obtained in the presence of DMSO. The negative values on the y-axis for the heat flow implies an exothermic heat release in the DSC sample. The heat flow (mW/mg) is plotted along the y-axis and the sub-zero temperatures (°C) are plotted along the top x-axis and time (s) is plotted on the bottom x-axis.

Fig. 2

Left, representative scanning electron micrographs (SEM) of sperm from X. helleri showing the relationship of sperm head and tail in alignment with model used to calculate the cell volumes and surface areas shown on the right. Right, the “geometric” model used to calculate the X. helleri sperm cell volumes and surface areas from the SEM images shown on the left.

Fig. 3

Volumetric response of X. helleri sperm cells as a function of sub-zero temperatures obtained using the DSC technique in the presence of extracellular ice (A), in the presence of extracellular ice and glycerol (B), and in the presence of extracellular ice and DMSO (C). The filled circles represent the experimentally obtained water transport (volumetric shrinkage) at a cooling rate of 20 °C/min. The dynamic cooling response at 20 °C/min is shown as a solid line and was obtained by using the “best fit” membrane permeability parameters (L_pg and E_Lp or L_pg[cpa] and E_Lp[cpa]) (Table 1) in the water transport equation (Eqs. (3) and (4)). The model-simulated equilibrium cooling response obtained is shown as a dotted line in all the figures. The non-dimensional cell volume is plotted along the y-axis and the sub-zero temperatures are shown along the x-axis. The error bars represent the standard deviation for the mean values of nine separate DSC experiments (n = 9).

Fig. 3

Volumetric response of X. helleri sperm cells as a function of sub-zero temperatures obtained using the DSC technique in the presence of extracellular ice (A), in the presence of extracellular ice and glycerol (B), and in the presence of extracellular ice and DMSO (C). The filled circles represent the experimentally obtained water transport (volumetric shrinkage) at a cooling rate of 20 °C/min. The dynamic cooling response at 20 °C/min is shown as a solid line and was obtained by using the “best fit” membrane permeability parameters (L_pg and E_Lp or L_pg[cpa] and E_Lp[cpa]) (Table 1) in the water transport equation (Eqs. (3) and (4)). The model-simulated equilibrium cooling response obtained is shown as a dotted line in all the figures. The non-dimensional cell volume is plotted along the y-axis and the sub-zero temperatures are shown along the x-axis. The error bars represent the standard deviation for the mean values of nine separate DSC experiments (n = 9).

Fig. 4

Contour plots of the goodness of fit parameter R² (= 0.96) for water transport response in X. helleri sperm cells in three different media: HBSS with no CPAs (A), HBSS with 14% glycerol (B), and HBSS with 10% DMSO (C). Note that the best-fit parameters (Table 2) are represented within the contours by a “#” (absence of CPAs), “+” (with 14% glycerol) and “*” (with 10% DMSO). The membrane permeability at 0 °C, L_pg (or L_pg[cpa]) (m³/Ns) is plotted on the y-axis while the apparent activation energy of the membrane, E_Lp (or E_Lp[cpa]) (kJ/mol) is plotted on the x-axis.

Fig. 5

Volumetric response of X. helleri sperm cells at various cooling rates as a function of sub-zero temperature using the best-fit membrane permeability parameters (Table 1). The changes in the normalized cell volume (V/V_o) are shown as a function of temperature for different cooling rates in X. helleri sperm cell suspensions without CPAs (A), with 14% glycerol (B), and with 10% DMSO (C). The water transport curves represent the model-simulated response for different cooling rates (from left to right: 5, 10, 20, 40, 60, 80, and 100 °C/min). The model-simulated equilibrium cooling response obtained is shown as a dotted line. The sub-zero temperatures are shown along the x-axis while the non-dimensional volume is plotted along the y-axis.

Fig. 5

Volumetric response of X. helleri sperm cells at various cooling rates as a function of sub-zero temperature using the best-fit membrane permeability parameters (Table 1). The changes in the normalized cell volume (V/V_o) are shown as a function of temperature for different cooling rates in X. helleri sperm cell suspensions without CPAs (A), with 14% glycerol (B), and with 10% DMSO (C). The water transport curves represent the model-simulated response for different cooling rates (from left to right: 5, 10, 20, 40, 60, 80, and 100 °C/min). The model-simulated equilibrium cooling response obtained is shown as a dotted line. The sub-zero temperatures are shown along the x-axis while the non-dimensional volume is plotted along the y-axis.

Fig. 6

Contour plots of the goodness of fit parameter R² (= 0.96) for water transport response in X. helleri sperm cells in three different media: HBSS with no CPAs (A), HBSS with 14% glycerol (B), and HBSS with 10% DMSO (C). Within each figure, the three contour correspond to the three assumed values of osmotically inactive cell volume, V_b (0.4V_o, 0.6V_o, and 0.8V_o). Note that the best-fit parameters (Tables 2 and 3) are represented within the contours by a “#” (V_b = 0.4V_o), “+” (V_b = 0.6V_o) and “*” (V_b = 0.8V_o). The membrane permeability at 0 °C, L_pg (or L_pg[cpa]) (m³/Ns) is plotted on the y-axis while the apparent activation energy of the membrane, E_Lp (or E_Lp[cpa]) (kJ/mol) is plotted on the x-axis.

Fig. 6

Contour plots of the goodness of fit parameter R² (= 0.96) for water transport response in X. helleri sperm cells in three different media: HBSS with no CPAs (A), HBSS with 14% glycerol (B), and HBSS with 10% DMSO (C). Within each figure, the three contour correspond to the three assumed values of osmotically inactive cell volume, V_b (0.4V_o, 0.6V_o, and 0.8V_o). Note that the best-fit parameters (Tables 2 and 3) are represented within the contours by a “#” (V_b = 0.4V_o), “+” (V_b = 0.6V_o) and “*” (V_b = 0.8V_o). The membrane permeability at 0 °C, L_pg (or L_pg[cpa]) (m³/Ns) is plotted on the y-axis while the apparent activation energy of the membrane, E_Lp (or E_Lp[cpa]) (kJ/mol) is plotted on the x-axis.