Experiment 4. Activation energy for boar spermatozoa glycerol and water permeability in the presence of extender. One hundred microliters of 2 M glycerol were added drop by drop over 60 sec to 100 jjlI of cell suspension diluted in Modena extender, yielding a final CPA concentration of 1 M glycerol. Cells were allowed to equilibrate for approximately 3 min before the 200 |xl was returned to isosmotic media. Changes in cell volume were measured over time. A total of 3 donors (n = 3) were used, and each experimental run was replicated three times for each donor. The experiments were performed at 22°C, 8°C, and 0°C in the presence of glycerol and EG, and at 22°C for DMSO.
Experiment 5. Simulation of cryoprotectant addition and removal. Computer simulations of glycerol and EG addition and removal to and from boar spermatozoa were performed using methods described by Gilmore et al. buy flovent inhaler
Experiment 6. Simulation of intracellular water volume flux during cooling and warming. Computer simulations of intracellular water volume flux during the cooling and warming of boar spermatozoa were performed using methods described by Liu et al.. The theoretical predictions of water loss during cooling and wanning were based upon the determined cryobiological parameters of boar spermatozoa and made the following assumptions: 1) above-zero activation energies were extrapolated to subzero temperatures, 2) a generic phase diagram was used for both glycerol and EG, and 3) the predictions assumed that optimal methods for CPA addition and removal were used. If any of these assumptions are incorrect, the results could potentially increase cell loss.