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.

Production of F1Offspring with Vitrified Sperm from a Live-Bearing Fish, the Green SwordtailXiphophorus hellerii

“…57 Previous studies in X. hellerii achieved cooling rates of 200°C/min by the use of a differential scanning calorimeter (DSC) but caused the sperm cells to lyse and become osmotically inactive. 58 In the same study, the optimal cooling rate predicted for samples without cryoprotectant was 90°C/min. In a different study, minimal post-thaw motility ( < 1%) was reported when 0.25-mL straws containing 80 lL samples of X. hellerii sperm with 14% Gly were plunged into liquid nitrogen.…”

“…70 Fish sperm, in contrast are characterized by low protein concentrations, containing mainly mineral compounds and low concentrations of other organic substances such as sugars. 71 Xiphophorus sperm 58 are similar in size, 72 with a head length of 3.6 lm (human 4-5 lm), midpiece length of 6.8 lm (human 7-8 lm), and flagellum length of 43 lm (human 45 lm). However Xiphophorus sperm have a higher surface area-to-volume ratio than human sperm (6.81 for Xiphophorus and 4.8 for humans) and a higher osmotically inactive cell volume than humans (0.6 for Xiphophorus and 0.5 for humans).…”

“…52 This fast warming rate could explain results from a previous report in X. hellerii in which samples were cooled at a relatively fast rate (200°C/ min) but the sperm were destroyed due to the slow warming rate used (20°C/min). 58 In addition, X. hellerii sperm in 0.25-mL straws that were plunged into liquid nitrogen (cooling of 1700°C to 2500°C/min) and thawed for 7 s in a 40°C water bath (thawing of around 1300°C to 2500°C/min) yielded minimal motility ( < 1%). 30 The reason for this could be the use of a slow warming rate compared with the rate suggested recently (118,000°C/min 52 ).…”

“…63 The intracellular nucleation temperature inferred by DSC for X. hellerii was -30°C. 58 In a similar live-bearing fish (Poecilia reticulata) the nucleation temperature was calculated to be in the range of -25°C to -32°C at cooling rates from 5°C/min to 100°C/ min. 64 It has been suggested that survival of vitrified sperm could be due to the presence of large amounts of osmotically inactive water bound to macromolecular structures, such as DNA and histones, or the presence of high molecular weight components in sperm that affect the viscosity and glass transition temperature of the intracellular cytosol.…”

“…However Xiphophorus sperm have a higher surface area-to-volume ratio than human sperm (6.81 for Xiphophorus and 4.8 for humans) and a higher osmotically inactive cell volume than humans (0.6 for Xiphophorus and 0.5 for humans). 58,73 These characteristics should allow the sperm from Xiphophorus to have higher hydraulic conductivity (water loss), higher cryoprotectant permeability, and in theory better cryopreservation success. Based on the membrane integrity results in the present study, we suggest that the plasma membrane is damaged either by intracellular ice formation 58 or by changes in the physical properties of the extracellular environment.…”