Effect of glycerol and cholesterol-loaded cyclodextrin on freezing-induced water loss in bovine spermatozoa

Recent experimental data show that incubating bovine sperm with cholesterol-loaded cyclodextrin (CLC) before cryopreservation increases the percentages of motile and viable cells recovered after freezing and thawing, compared with control sperm. In the present study, we report the effect of incubating bovine sperm with CLC on the subzero water transport response and the membrane permeability parameters (reference membrane permeability (L_pg) and activation energy (E_Lp)). Water transport data during freezing of bovine sperm cell suspensions were obtained at a cooling rate of 20 °C/min under three different conditions: 1. in the absence of cryoprotective agents (CPAs); 2. in the presence of 0.7 M glycerol; and 3. in the presence of 1.5 mg/ml CLC and 0.7 M glycerol. With previously published values, the bovine sperm cell was modeled as a cylinder of length 39.8 μm and radius 0.4 μm, with osmotically inactive cell volume (V_b) of 0.61 V_o, where V_o is the isotonic cell volume. By fitting a model of water transport to the experimentally obtained data, the best-fit water transport parameters (L_pg and E_Lp) were determined. The predicted best-fit permeability parameters ranged from L_pg = 0.02 to 0.036 μm/min-atm and E_LP = 26.4 to 42.1 kcal/mol. These subzero water transport parameters are significantly different from the suprazero membrane permeability values (obtained in the absence of extracellular ice) reported in the literature. Calculations made of the theoretical response of bovine spermatozoa at subzero temperatures suggest that the optimal cooling rate to cryopreserve bovine spermatozoa is 45-60 °C/min, agreeing quite closely with experimentally determined rates of freezing bovine spermatozoa.