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

The hypoosmotic swelling test (HOS) is one of the methods used to evaluate sperm quality in mammals. This test is based on the swelling ability that functional spermatozoa have when submitted to hypoosmotic solutions. Only a slight increase in size is caused in rainbow trout spermatozoa in such conditions and it is not possible to distinguish between reactive cells (cells who were capable to increase in volume) and non-reactive cells (did not increase in volume) under light microscopy. In our approach we have used the coulter counter to verify the effectiveness of the HOS test in this species. Semen was diluted in different hypoosmotic solutions (50, 100, 150, 200, 250 and 320 mosM/kg) and cell volume measured at different times after dilution (30 s, 2, 5, 10, 20, and 30 min). The higher percentage of reactive 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 caused the overlapping of 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 rates of live/dead cells. Two parameters were analysed after swelling: the increase in volume and the percentage of cells over a standard 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 fact suggests that HOS test could be used to analyse the integrity and functionality of rainbow trout fresh sperm. To study the reliability of this test in cryopreserved sperm, simple linear regressions were made between cell viability determined by Hoechst 33285 dye and the two parameters obtained from coulter counter data. No significant correlation was observed in either case, showing that structural and functional integrity do not correlate after freeze/thaw. Consistently, the HOS test is not a reliable method to evaluate cryopreserved sperm quality in rainbow trout.