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 f unctional spermatozoa have when submitted to hypoosmotic solutions. Only a slight increase in size is caused in rainbow bout spermatozoa in such condi tions and it is not possible to distinguish between reactive cells (cells w ho were capable to increase in volume) and non-reactive cells (did not incr ease in volume) under light microscopy. In our approach we have used the co ulter counter to verify the effectiveness of the HOS test in this species. Semen was diluted in different hypoosmotic solutions (50, 100, 150, 200, 25 0 and 320 mosM/kg) and cell volume measured at different times after diluti on (30 s, 2, 5, 10, 20, and 30 min). The higher percentage of reactive cell s was achieved with the 100 mosM/kg solution and swelling occurred before 3 0 s. Even with this solution, the small increase in cell size caused the ov erlapping of volumes from swollen and non-swollen spermatozoa. In order to analyse the data and to choose a parameter suitable for assessing cell reac tivity, the test was performed in samples containing known rates of live/de ad cells. Two parameters were analysed after swelling: the increase in volu me and the percentage of cells over a standard volume (reactive cells). Res ults showed a high correlation between the percentages of reactive cells an d the known rate of live cells (r(2) = 0.65). This fact suggests that HOS t est could be used to analyse the integrity and functionality of rainbow tro ut fresh sperm. To study the reliability of this test in cryopreserved sper m, simple linear regressions were made between cell viability determined by Hoechst 33285 dye and the two parameters obtained from coulter counter dat a. No significant correlation was observed in either case, showing that str uctural and functional integrity do not correlate after freeze/thaw. Consis tently, the HOS test is not a reliable method to evaluate cryopreserved spe rm quality in rainbow trout. (C) 1999 Elsevier Science B.V. All rights rese rved.