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The honey bee, Apis mellifera, is one of very few insects for which artificial insemination is possible, and preservation of semen has been attempted. Honey bee queens normally mate with 7–20 males early in life, store the semen in the spermatheca and release approximately 30 sperm to fertilize each egg. Fertilized eggs are females (queens or workers) and unfertilized eggs are males (drones). The queen controls release of spermatozoa, laying only worker eggs across large areas of comb, and drone eggs in small separate groups. As stored spermatozoa are depleted, a queen will begin to lay mixed groups of workers and drones. Semen cryopreserved following existing protocols (Harbo JR 1983 Annals Entomol. Soc. Amer. 76, 890–891) has less than 20–25% live spermatozoa (based on percentage of workers reared). This viability level is insufficient to successfully fertilize all of the 1000–1500 eggs per day from an active queen, and the colony slowly dwindles and dies. Using dual fluorescent staining (Collins AM and Donoglye AM 1999 Theriogenology 51, 1513–1523). I have determined that semen with 46% live spermatozoa (50% fresh and 50% freeze-killed semen v/v) or more, produces consistent laying patterns with all fertilized eggs (workers) from inseminated queens (Collins AM 2000 Apidologie 31, 421–429). The current study was done to determine how long queens inseminated with mixed fresh:frozen semen would continue to produce normal brood (collective term for all eggs, larvae and pupae), as compared to those inseminated with all fresh semen, or naturally mated in flight. In spring, sister queens were reared and inseminated with [1] all fresh semen, [2] half fresh and half freeze-killed semen, or [3] ¼ fresh and ¾ freeze-killed semen, or [4] were allowed to mate naturally. The queens were evaluated monthly in individual colonies for percentage of worker offspring v. drone offspring and area of comb with eggs, larvae and pupae, until the queen failed. As expected, the inseminated queens produced less brood than did the naturally mated [4] queens. All of the queens with only fresh semen [1] produced normal brood with 100% workers, and lived well into the winter. Eighty-eight percent of the queens inseminated with half freeze-killed semen [2] performed as well, although some of those failed within a few months. The remaining 22% began laying with mixed worker and drone brood. In treatment [3], 60% of the queens produced variable but high levels of drones in reduced areas of egg laying. Those queens in this group that did lay well (40%) also survived into the winter. These results mean that preserved semen that has 50% or better viable sperm has a good probability of producing inseminated queens that will lay normally for a complete beekeeping season. This is sufficient time for breeders to rear daughter queens from such matings, and incorporate desirable genotypes into a breeding program.

90EFFECTIVE VIABILITY THRESHOLD FOR PRESERVED HONEY BEE SEMEN