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Methods of storage of honeybee semen: evaluation of honeybee spermatozoa viability with flow cytometry.
Savvulidi, Filipp ; Titěra, Dalibor (advisor) ; Jan, Jan (referee)
Assessment of semen quality (sperm viability) is an essential for the efficient honeybee semen storage. The motility of spermatozoa is not the ultimate parameter of sperm viability. Instead, the viability of spermatozoa is assessed by testing their structural or functional plasma membrane integrity. These assays are based primarily on the technique of microscopy evaluation of either fluorescent DNA dyes or hypo-osmotic "tail-coiling" reaction of spermatozoa. However, the technique of microscopy evaluation is time-consuming, subjective and often is not accurate. On the other hand, the technique of flow cytometry is rapid, objective and precise. Nowadays, this technique is affordable for many institutions, as the price of "benchtop" flow cytometer is relatively low. To best of our knowledge, there is currently no flow cytometry assay available for evaluation of functional integrity of honeybee spermatozoa. Here we report the establishing of novel flow cytometry assay for simultaneous evaluation of structural and functional plasma membrane integrity. DRAQ7, the new fluorescent DNA dye was used to test the structural integrity of plasma membranes. The hypo-osmotic "tail-coiling" reaction was used for flow cytometric assessment of functional integrity of spermatozoa plasma membranes. The combination of both is a valuable tool for rapid and precise evaluation of sperm viability in honey bees.
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