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This report is about cryopreservation of honey bee semen. There has been little advancement of this technology over the past 20 years. Cytotoxicity of the cryoprotectants, temperature sensitivity, freezing rate, and cold shock were investigated. The least toxic cryoprotectant was DMSO. Spermatozoa were tolerant to temperatures up to 40 • C. A programmable freezing rate of 3 • C/min proved superior in most treatments when compared to a freezing rate of approximately 28 000 • C/min. Highest viability of spermatozoa (93%) post cryopreservation resulted from the treatment containing a 10% DMSO diluent, slow cooled to just above freezing, and frozen at a rate of 3 • C/min. Spermatozoa frozen in such a manner yielded viability and motility indistinguishable from that of unfrozen semen. Promising results warrant a field study. cryopreservation / Apis mellifera / spermatozoa / cold shock / vitrification

Much of the world's food production is dependent on honey bees for pollination, and expanding food production will further increase the demand for managed pollination services. Apiculturists outside the native range of the honey bee, in the Americas, Australia and eastern Asia, have used only a few of the 27 described subspecies of honey bees (Apis mellifera) for beekeeping purposes. Within the endemic ranges of a particular subspecies, hybridisation can threaten native subspecies when local beekeepers import and propagate non-native honey bees. For many threatened species, cryopreserved germplasm can provide a resource for the preservation of diversity and recovery of endangered populations. However, although instrumental insemination of queen honey bees is well established, the absence of an effective means to cryopreserve honey bee semen has limited the success of efforts to preserve genetic diversity within the species or to develop repositories of honey bee germplasm for breeding purposes. Herein we report that some queens inseminated with cryopreserved semen were capable of producing a substantial number of fertilised offspring. These diploid female larvae were used to produce two additional sequential generations of new queens, which were then back-crossed to the same stock of frozen semen. Our results demonstrate the ability to produce queens using cryopreserved honey bee spermatozoa and the potential for the establishment of a honey bee genetic repository.