Thermal characterization of Nakagata's mouse sperm freezing protocol

This paper reports the results of an experimental study of the warming and cooling rates achieved using the popular Nakagata Protocol for murine sperm cryopreservation. Problems with the storage and maintenance of the huge number of genetically engineered mouse strains have led to an increased need for murine sperm preservation. Recent studies have begun to focus on optimizing the cryopreservation of murine sperm by carefully studying the effects of cooling and warming rates on sperm survival. In current practice, however, the Nakagata protocol is widely used. The actual cooling and warming rates achieved using the Nakagata protocol have not previously been determined; and the Nakagata protocol has a number of unspecified parameters which we have found can significantly affect cooling rates, warming rates and sperm survival. A detailed study of the thermal response of samples frozen and thawed using the Nakagata protocol reveals that the cooling rates range from 30 to almost 300°C per minute depending on the exact manner in which the Nakagata protocol is implemented. Warming rates range from 160°C/min to about 1000°C/min. Sperm survival depended significantly on the particular cooling rate achieved, and less strongly on the warming rates. Overall, it was found that the particular manner in which the Nakagata protocol was implemented could strongly affect cooling rates and sperm survival; and, consistent with the findings of Mazur and Koshimoto, an optimal cooling rate appears to exist in the range of cooling rates that can be achieved using the Nakagata protocol.