Abstract
Cryopreservation of honeybee (Apis melifera) drone semen remains a significant challenge. Despite decades of research, maintaining sperm viability and functionality after thawing continues to limit the practical use of frozen semen. This study investigated the effect of post-thaw dilution of cryopreserved drone semen with Bee Semen Solution (BSS) on semen viability and queen reproductive performance. Queen bees were instrumentally inseminated with fresh or cryopreserved semen, either undiluted or diluted with BSS after thawing. The impact of these treatments on the number and quality of spermatozoa in the spermatheca and on brood pattern was assessed. Cryopreservation markedly affected sperm quality and the reproductive performance of inseminated queens, resulting in lower sperm viability and reduced fertility compared with fresh semen. However, post-thaw dilution of semen with BSS tended to improve sperm parameters in the spermatheca and queen fertility. The results indicate that post-thaw handling of semen, including the use of appropriate diluents, may play an important role in improving the outcomes of insemination with dialysis method cryopreserved semen. We also emphasize that multiple insemination should be considered as a method of improving spermatozoa number in spermahteca and, consequently, queen fertility.