The effect of the osmolality of sugar-containing media, the type of sugar, and the mass and molar concentration of sugar on the survival of frozen-thawed mouse sperm.

Several factors have contributed to problems in mouse sperm cryopreservation, and we and others have found ways to ameliorate them. These include high sensitivity to several types of mechanical stresses and to oxygen-derived free radicals, low tolerance to osmotic cell volume changes, and rather rigorous requirements for cooling and warming rates. Another important factor is the cryoprotective agent. Mouse sperm are unusual in that our best results have been obtained in media containing the nonpermeating sugar raffinose (18% w/v) and lacking glycerol. This paper deals with questions about the basis of the protective action of sugars, and whether raffinose is unusual or unique in its ability to confer protection. More specifically, we investigated whether protection was more related to the total osmolality of the freezing solution, to the mass concentration of sugar, or to the molarity of the sugar, and we looked to see whether there are effects attributable to specific sugars. To investigate these questions, mouse sperm were frozen at the optimal rate of 25 degrees C/min in solutions prepared with different proportions of three sugars-raffinose, sucrose, and glucose-dissolved in 1/4x PBS. In the first experimental series, the total osmolality and the total sugar molarity were varied from 400 to 700 mOsm and from 300 to 530 mM, respectively, while holding the mass concentration of sugar constant at 18% (w/v). In the second experimental series, the mass concentration of sugars was varied from 10 to 18% while the sugar molarity and solution osmolality remained constant at 300 mM and 420 mOsm, respectively. The results suggest that protection against freezing and thawing depends more on the mass concentration of the sugar than on its molar concentration, a conclusion that has mechanistic implications.