Preliminary experiments for the genotyping of domestic bee (Apis mellifera) and sperm freezing

Abstract

We started to chart the genetic diversity and purity of the Carpathian honeybee substance by using genomic DNA markers. In different families, genotypes of queen bee can be determined by using STR markers and we can choose which the best for long-term ex-situ storage is. We can use the frozen-thawed semen for artificial insemination of the queen bees and restore or use the stored lines in directional crossings. Therefore, we must devise a new, simply method for the extraction and frozen of the semen. We have developed a stereo-microscopic method of preparation; the gametes were obtained from the seminal vesicle. The medium containing the sperm was centrifuged at 700g for 2 min, the supernatant was removed and the precipitate diluted to a specific amount and mixed with cryoprotectant agents (CPA) 3: 2: DMSO25%+BSS+egg yolk; DMSO10%+BSS+egg yolk, DMSO25%+Harbo+egg yolk, DMSO10%+Harbo+egg yolk. We found that the DMSO mixture is non-toxic for sperm. 10μl of the semen mixed with CPA was filled into a 0.25μl of straw with 80μl of diluent and 10μl of CPA. After incubation on ice, one half of the samples were placed directly in the liquid nitrogen container and the other half was cooled to -40°C with a programmable freezer at 3°C/min. The thawing was done in a 38.5°C water bath. The samples were incubated at 38.5°C for 10 minutes and then the motility was re-examined by phase contrast microscopy. Semen mixed with 10% DMSO solution were killed by freezing, while 80% of sperm mixed with 25% DMSO solution frozen by programmed slow cooling survived the treatment but exhibited a lower motility after thawing.