Effect of osmolality and oxygen tension on the survival of mouse sperm frozen to various temperatures in various concentrations of glycerol and raffinose

Cryopreserved mouse sperm are beginning to be used to meet the demand of a reliable cost-effective method for maintaining the rapidly expanding number s of lines of mutant mice. However, successful and reproducible cryopreserv ation has proven to be a difficult problem. Furthermore, the underlying fac tors responsible for success or failure are mostly obscure. Several contrib utors to these difficulties have been identified. Our laboratory has found that mouse sperm are extremely susceptible to the mechanical stresses assoc iated with pipetting. mixing, and centrifugation, and others have found tha t they are severely limited in their tolerance to osmotic volume changes. W e have hypothesized two other contributors to the difficulties. One is that the concentrations of glycerol used in published protocols are substantial ly lower than those found to be optimal for most mammalian cells. The other hypothesis relates to the fact that mouse sperm membranes are especially s usceptible to damage from oxygen-derived free radicals. That damage may red uce their ability to survive freezing. If so, survival ought to increase if the concentration of oxygen is kept low throughout the procedure. To achie ve low levels, we have incorporated an Escherichia coli membrane fraction, Oxyrase, into all media. A previous report showed a protective effect. That is confirmed here under a broader range of conditions. The conditions stud ied have been the individual and interactive effects of the concentrations of glycerol, raffinose, and phosphate-buffered saline (PBS) on motility aft er freezing at 21 degreesC/min to -70 degreesC. Cryoprotection increased wi th increasing raffinose concentration. provided that the concentration of P BS was appropriately reduced to hold the total osmolality of nonpermeating solutes to within tolerated limits. Surprisingly, the beat results were ach ieved in the total absence of glycerol. The highest motilities to date (68 +/- 8%) after freezing to -70 degreesC have been achieved using media conta ining Oxyrase, 0 M glycerol, and 18% raffinose in 1/4 x strength modified P BS. We also determined the motility loss after freezing to intermediate tem peratures, i.e., -10 and -30 degreesC. The major motility loss occurred by - 10 degreesC, especially in the absence of Oxyrase. These results suggest that a major problem in the freezing of mouse sperm is the physical stress resulting from extracellular ice crystal formation. Oxyrase appears to less en that damage substantially. (C) 2000 Academic Press.