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

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 °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 μm in length and 0.59 μm in diameter with an osmotically inactive cell volume (Vb) of 0.6Vo, where Vo 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, Lpg or Lpg[cpa] and the activation energy, ELp or ELp[cpa]) of the Xiphophorus helleri sperm cell membrane were determined. The best-fit membrane permeability parameters at 20 °C/min in the absence of CPAs were: Lpg = 0.776 × 10−15 m3/Ns (0.0046 μm/min atm), and ELp = 50.1 kJ/mol (11.97 kcal/mol) (R2 = 0.997). The corresponding parameters in the presence of 14% glycerol were Lpg[cpa] = 1.063 × 10−15 m3/Ns (0.0063 μm/min atm), and ELp[cpa] = 83.81 kJ/mol (20.04 kcal/mol) (R2 = 0.997). The parameters in the presence of 10% DMSO were Lpg[cpa] = 1.4 × 10−15 m3/Ns (0.0083 μm/min atm), and ELp[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 °C/min and were in close agreement with recently published, empirically determined optimal cooling rates.