Use of different semen volumes in hypoosmotic test of adult Nelore bulls.

The objective of this study was to evaluate different proportions of semen: hypoosmotic solution in the hypoosmotic swelling test and their relationship with semen freezability in Zebu bulls. A total of 15 ejaculates from three adult Nelore bulls were used. Physical and morphological features were analyzed in fresh semen, as well as supravital staining and hypoosmotic swelling test. In the hypoosmotic test, a hypoosmotic solution with 100 mOsm/kg osmolality using 15 minutes incubation at 37 °C was used both in fresh and frozen/thawed semen. Four semen volumes in 1-ml hyposmotic solution were used: 10, 20, 50 and 100 μL. Cryopreserved samples were thawed and submitted to hypoosmotic tests, supravital staining, slow thermo-resistance test and fluorescent staining. Mean values and standard deviations of the percentage of reactive sperm cells in the hypoosmotic test in fresh and frozen/thawed semen were 69.3 ± 11.8 and 20.5 ± 6.8, respectively. There was no correlation between the hypoosmotic test and physical and morphological features and the complementary tests performed on fresh and frozen/thawed semen. None of the plasma membrane integrity tests was able to predict bull semen freezability. It can be concluded that the hypoosmotic test can be performed with 20 to 100 μL fresh semen, and 10 to 100 μL of frozen/ thawed semen in 1 mL of hypoosmotic solution without interfering with their results, but 100 μL should be used in both, because it considerably improves the view of the slides.