The use of the hypo-osmotic swelling test, water test, and supravital staining in the evaluation of drone sperm

Abstract

The aim of the present study was to investigate the suitability of the water test and hypo-osmotic swelling (HOS) test for analyzing honeybee semen. In particular, the relationships between these tests of the integrity of the sperm plasma membrane and tests of sperm motility and sperm viability were measured using SYBR-14/PI. To this end, semen was obtained from mature drones (16 days or older) collected from four colonies. The means of the per-drone sperm concentration, sperm motility, and live spermatozoa were 1.47 × 106, 76.0%, and 87.2%, respectively. The percentage of spermatozoa with swollen tails was 92.2% as measured with the water test. With the HOS tests at 50, 100, and 150 mOsm/kg, the percentages of spermatozoa with swollen tails were 94.2%, 90.5%, and 85.6%, respectively, after a 30-min incubation and 92.2%, 90.5%, and 88.0%, respectively, after a 60-min incubation. It was observed that subjecting honeybee spermatozoa to a hypo-osmotic solution resulted in clearly identifiable swollen-tail spermatozoa. The percentages of swollen-tail spermatozoa obtained using the water and HOS tests were higher than the percentages of viable and motile spermatozoa. Similar results were obtained using the water test and the HOS test at 50 mOsm/kg. It was concluded that HOS and water tests are suitable and simple measures of the functional integrity of the plasma membranes of bee spermatozoa, and they may be useful additions to standard methods of semen evaluation in mammals.