Genetically engineered mouse (GEM) and rat (GER) lines are excellent animal models for many important human diseases (Cox, 2003;Green and Hudson, 2005). However, maintenance of these lines by standard breeding techniques is producing unsustainable pressure on facilities and budgets. Consequently, there is a critical need to develop reliable and cost-effective alternative means of rodent germplasm preservation. Sperm cryopreservation would be a simpler and substantially less costly approach compared to embryo cryopreservation which is currently standard (Thornton, 1999), provided that sufficient numbers of sperm survive freezing and thawing in a reliable manner. However, mouse sperm survival rates after cryopreservation are highly variable among different strains (Mazur et al., 2000;Critser and Mobraaten, 2000;Yildiz et al., 2007). For rat sperm, to date there is only one group that has reported successful, repeatable cryopreservation (Nakatsukasa et al., 2001, 2003) and the survival rate was extremely low (<10%). Rodent sperm have an unusual morphology and are also extremely mechanically sensitive (Katkov and Mazur, 1998;Mazur et al., 1998;Nakatsukasa et al., 2001;Si et al., 2006;Walters et al., 2005). Related to this mechanical sensitivity, mouse sperm are vulnerable to damage associated with the morphology of the extracellular ice formed during cryopreservation. However, current paradigms for developing cryopreservation strategies focus on factors such as the formation of intracellular ice and the high intra/extracellular solute concentrations during freezing, rather than the mechanical sensitivity of cells or ice-cell interactions (Mazur 1977, 1984;Muldrew and McGann 1994). Therefore, novel techniques to prevent extracellular ice damage should be applied to improve rodent sperm survival rates after cryopreservation. It is therefore the overall goal of this proposal to establish a rodent sperm cryopreservation approach to prevent the mechanical damage to cells associated with extracellular ice formation. To that end, a new device will be produced to generate a directional growth of relatively large and smooth ice crystals in the cryoprotectant solutions and orientate sperm tails in the same direction of the ice growth. This will be achieved by applying the dielectrophoretic orientation and directional solidification techniques with electrothermal controls. Production of the proposed device will entail: (1) production of a directional solidification stage with embedded Peltier electrodes to accurately control the temperature gradient and ice morphology and maintain ice growth direction;(2) Production of an operational chip with ice growth channels which will also contain cells and can be loaded on the directional solidification stage;and (3) Production of a dielectrophoretic orientation subsystem to provide a proper electric field gradient and magnitude inside the ice growth channels so that sperm cells can be orientated in the same direction as the ice growth via the dielectrophoretic force. In this way, the compressive/tensile and shearing stresses exerted by ice crystals on sperm tails can be diminished to prevent their mechanical damage. Ultimate feasibility of this proposed novel system will be considered achieved if 60% post-thaw mouse and rat sperm motility is achieved. In Phase II, the device will be further optimized and developed for ease of end use and production.
Genetically engineered mouse (GEM) and rat (GER) lines are very useful models for many important human diseases;however maintaining these animals by standard breeding techniques is very difficult and expensive. Consequently, there is a critical need to develop reliable and cost-effective means of preserving mouse and rat genetic information. Sperm cryopreservation would be a simpler and substantially less costly approach compared to embryo cryopreservation which is currently the standard approach. This proposed project would optimize a device and method for effective freezing of mouse and rat sperm from these valuable research animals.
