Successful Cryoprotectant‐Free Vitrification of Honey Bee (Apis mellifera) Drone Sperm With Royal Jelly Supplemented Extender

ABSTRACT In trying to protect honey bee species and maintain genetic diversity, employing effective approaches for drone sperm conservation is crucial. Based on literature, drone sperm cryopreservation extenders and methods have not been fully optimized. Our research aim was to enhance drone bee sperm preservation by incorporating royal jelly (RJ) in the extender of the cryoprotectant‐free vitrification method. Drone sperm was collected from adult drone bees (36 days old) using the manual inversion method. Different concentrations of RJ (0%, 0.5%, 1%, 2.5% and 5%) were added to the extender. Diluted sperm were cryopreserved using a cryoprotectant‐free vitrification technique where 10 µL aliquots of the diluted sperm were directly dropped into the liquid nitrogen and then stored. Data were analysed based on a completely randomized design with ten replications. Sperm quality parameters, including motility, viability and DNA damage, were evaluated in vitro. Queens were artificially inseminated to measure the ability of motile sperm to reach the spermathecae. The results showed that 1% and 2.5% of RJ supplementation significantly enhanced sperm motility and viability and reduced DNA fragmentation compared to control and higher RJ concentrations. Specifically, the 1% RJ group resulted in the highest sperm viability, while both the 1% and 2.5% groups maintained lower DNA fragmentation rates. Queens inseminated with sperm treated with 1% and 2.5% RJ showed a notably higher number of motile sperm in their spermathecae. In conclusion, supplementation of 1% RJ to the cryoprotectant‐free vitrification media may improve drone sperm quality parameters post‐warming. Our findings provide valuable insights into optimizing drone bee sperm preservation, contributing to the conservation of these vital pollinators.