The hypoosmotic swelling test in fresh rabbit spermatozoa

The hypoosmotic swelling test (HOST) has proved to be a good tool for evaluating the membrane integrity of spermatozoa of various domestic animals including cattle, horses, and swine. However, the best approach for using this technique in rabbit semen has not been tested. The present study aimed to establish the best hypoosmotic solution (HS) for testing membrane integrity in fresh rabbit semen. Sucrose solutions with the following osmolarities were used: 50, 60, 75, 100, 125 and 150 mOsm/L. Semen samples (n = 30) were collected from five mature White New Zealand rabbits (six collections per rabbit) at 72 h intervals. After macroscopic evaluation, 10 μL of semen was immediately added to 2 mL of each solution and incubated for 1 h at 37 °C. Sequentially, 20 μL of semen diluted in HS were evaluated with oil immersion using a phase-contrast microscope. A total of 200 spermatozoa were counted in at least five different fields, and sperm tails were classified as non-coiled, coiled, and strongly coiled. The respective percentages of spermatozoa with coiled tails (coiled plus strongly coiled) in the six solutions listed above were 54.8, 65.2, 54.3, 53.9, 38.9 and 29.4%. Percentage of strongly coiled spermatozoa was: 40.2, 51.0, 43.2, 41.5, 32.7 and 26.9 for the six solutions, respectively. According to total and strong coiling 60 mOsm/L was superior to others treatments (P < 0.05). Results suggest that the 60 mOsm/L solution would be most desirable for use in HOST in fresh rabbit spermatozoa.