Comparison of glycerol, other polyols, trehalose, and raffinose to provide a defined cryoprotectant medium for mouse...

Most procedures for mouse sperm cryopreservation have utilized raffinose to provide hypertonicity for cell desiccation prior to freezing and glycerol to block intracellular ice formation. Trehalose has been shown in other cell systems to provide positive protection to the plasma membrane and so was examined as a replacement for raffinose. Comparison of 3 and 6% glycerol and 7.5 and 20% sugar showed that 6% glycerol and 7.5% sugar gave maximal protection consistently and so were adopted as standard. Comparison of raffinose and trehalose at this concentration showed trehalose to give significantly better recovery of intact cells: 48 +/- 6% for trehalose, 36 +/- 9% for raffinose (+/- SE, n = 5; arc sine transformed data; P < 0.01). Less hydrophilic polyols should prove more permeant to the membrane than glycerol, enter the cell rapidly, and so possibly inhibit lethal intracellular ice formation effectively. We hypothesized that one of these polyols plus glycerol would be a more effective cryoprotectant than glycerol alone. The polyols tested as supplements to 6% glycerol were propane-1,2-diol, propane-1,3-diol, 1,1,1-tris-(hydroxymethyl)ethane (THME), and 2-ethyl-2-(hydroxymethyl)-propane-1,3-diol (EHMP). With 6% glycerol and 7.5% raffinose or trehalose, the two diols and THME gave less cryoprotection than with glycerol alone, and EHMP reduced postthaw membrane integrity to nil, thus invalidating the hypothesis. Comparison of bicarbonate-containing medium MJB to bicarbonate-free medium NTP, both with 6% glycerol/7.5% trehalose, showed no difference in recovery of membrane-intact cells. For ease of pH maintenance, NTP was chosen for studies of addition prefreeze and removal postthaw of 6% glycerol/7.5% trehalose cryoprotectant with in vitro fertilization as endpoint. Three protocols for cryoprotectant handling were tested: serial addition/dilution; dialysis addition and removal; and dialysis addition and direct insemination without cryoprotectant removal. The last proved significantly superior (P < 0.01), giving 62% fertilized eggs, normalized to controls, compared to 21% for dialysis addition and removal and 32% for serial addition and dilution. The glycerol/trehalose combination thus provides a defined cryoprotectant which, when used with addition by dialysis prefreeze and direct insemination postthaw, yields a satisfactory yield of fertilized eggs in an in vitro fertilization system.