Development of a honey bee (Apis mellifera) semen extender

Decimation of honey bee populations necessitates advancement of artificial breeding technology. The ability to transport, store, and utilize germplasm from flourishing breeding stocks is essential. The beekeeping industry has a well-developed technique for instrumental insemination that can be utilized for selective breeding. However, the development of a suitable diluent for the storage and transport of semen are inadequate. Honey bee semen has the unique characteristic of remaining viable for days and possibly months if mixed with a properly formulated diluent. Organic molecules, including antibiotics that comprise a cell culture media have potential deleterious effects on cells when exposed to light. The time required to collect sufficient volumes of semen for multiple inseminations and the inseminations themselves means that the semen and diluent used in the collection process are exposed to light for extended periods. In developing our semen diluent we selected buffers and antibiotics based on their photostabiltity. Stability was determined by measuring singlet oxygen produced during exposure to florescent light or sunlight using the RNO+imidazole assay. Osmolality and pH of a cell culture solution are basic yet likely important factors to cell survival. Osmolality of spermathecal fluid and seminal plasma was measured using freezing point depression. Precautions were taken to avoid evaporation during collection of the fluid. Ion-selective microelectrodes were used to monitor pH of the semen/diluent mixture to verify pH stability over time. Previous reports on the constituents of seminal plasma were referenced so the formula would be a synthetic version of the natural plasma.