The hypoosmotic swelling test: Its employment as an assay to evaluate the functional integrity of the frozen-thawed bovine sperm membrane

The objective of this study was to determine the effectiveness of the hypoosmotic swelling (HOS) test together with the supravital test as a means of evaluating the functional integrity of frozen-thawed bovine sperm membrane. A solution consisting of equal parts of fructose and sodium citrate was prepared and the osmolality varied from 50 to 300 mOsm/L. From these various solutions under study, the 100 mOsm/L solution resulted in a maximal number of clearly identifiable swollen spermatozoa. The results from the supravital test indicated that the HOS solution preserved the integrity and prevented excessive lysis of the sperm membrane during the assay. A good correlation was found between the percentage of motile spermatozoa and spermatozoa that reacted to the HOS test (r = 0.73) and between the percentage of sperm with intact membranes and HOS reactive sperm (r = 0.81). Spermatozoa showing swelling of the entire tail region accounted for more than 60% of the total swelling for the HOS solution at 100 mOsm/L. The results obtained in this study indicate that frozen-thawed bovine spermatozoa did react to the HOS test. This technique could prove useful in studies involving the function of the sperm membrane and could possibly predict the sperm's ability to fertilize.