The hypoosmotic swelling test performed with Coulter counter: a method to assay functional integrity of sperm membrane in rainbow trout.

摘要

The hypo-osmotic swelling test (HOS) is based on the swelling ability that functional spermatozoa have when submitted to hypo-osmotic solutions. In rainbow trout (Oncorhynchus mykiss), the test produces only a slight increase in the size of the spermatozoa and it is not possible to distinguish between reactive cells (cells capable of increasing in volume) and non-reactive cells (cells that do not increase in volume) under light microscopy. In this study, a Coulter counter was used to verify the effectiveness of the HOS test in rainbow trout. Semen was diluted in hypo-osmotic solutions (50, 100, 150, 200, 250 and 320 mosm/kg) and cell volume was measured at different times after dilution (30 s and 2, 5, 10, 20 and 30 min). The highest percentage ofreactive cells was achieved with the 100 mosm/kg solution and swelling occurred before 30 s. Even with this solution, the small increase in cell size still produced overlapping volumes from swollen and non-swollen spermatozoa. In order to analyse the data and to choose a parameter suitable for assessing cell reactivity, the test was performed in samples containing known proportions of live/dead cells. Two parameters were analysed after swelling: the increase in volume and the percentage of cells over astandard volume (reactive cells). Results showed a high correlation between the percentages of reactive cells and the known rate of live cells (r2 = 0.65). This suggests that the HOS test could be used to analyse the integrity and functionality of freshspermatozoa of rainbow trout. To study the reliability of the test in cryopreserved spermatozoa, simple linear regressions were made between cell viability determined by Hoechst 33285 dye and the 2 parameters obtained from Coulter counter data. No significant correlation was observed in either case, showing that structural and functional integrity do not correlate after freezing and thawing. It is concluded that the HOS test is not a reliable method for the quality evaluation of cryopreserved spermatozoa in rainbow trout.