COMPARISON OF GLYCEROL, OTHER POLYOLS, TREHALOSE, AND RAFFINOSE TO PROVIDE A DEFINED CRYOPROTECTANT MEDIUM FOR MOUSE SPERM CRYOPRESERVATION

Most procedures for mouse sperm cryopreservation have utilized raffino se to provide hypertonicity for cell desiccation prior to freezing and glycerol to block intracellular ice formation. Trehalose has been sho wn in other cell systems to provide positive protection to the plasma membrane and so was examined as a replacement for raffinose. Compariso n of 3 and 6% glycerol and 7.5 and 20% sugar showed that 6% glycerol a nd 7.5% sugar gave maximal protection consistently and so were adopted as standard. Comparison of raffinose and trehalose at this concentrat ion showed trehalose to give significantly better recovery of intact c ells: 48 +/- 6% for trehalose, 36 +/- 9% for raffinose (+/- SE, n = 5; are sine transformed data; P < 0.01). Less hydrophilic polyols should prove more permeant to the membrane than glycerol, enter the cell rap idly, and so possibly inhibit lethal intracellular ice formation effec tively. We hypothesized that one of these polyols plus glycerol would be a more effective cryoprotectant than glycerol alone. The polyols te sted 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-(hydroxym ethyl)-propane- 1,3-diol (EHMP). With 6% glycerol and 7.5% raffinose o r 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 bicarbonatz-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 pr efreeze and removal postthaw of 6% glycerol/7.5% trehalose cryoprotect ant with in vitro fertilization as endpoint. Three protocols for cryop rotectant handling were rested: serial addition/dilution; dialysis add ition and removal; and dialysis addition and direct insemination witho ut cryoprotectant removal. The last proved significantly superior (P < 0.01), giving 62% fertilized eggs, normal ized to controls, compared to 21% for dialysis addition and removal and 32% for serial addition a nd dilution. The glycerol/trehalose combination thus provides a define d cryoprotectant which, when used with addition by dialysis prefreeze and direct insemination postthaw, yields a satisfactory yield of ferti lized eggs in an in vitro fertilization system. (C) 1998 Academic Pres s.