Assessing undiluted, diluted and frozen-thawed Nili-Ravi buffalo bull sperm by using standard semen assays

Hypo-osmotic swelling test (HOST), eosin-nigrosin staining and normal apical ridge test (NAR) were used to determine integrity of plasma membrane and acrosome of undiluted, diluted (cooled to 5oC) and frozen-thawed sperm. Semen from seven bulls was used. For diluted and frozen-thawed sperm, three doses were pooled at 37oC. Percentage motility was assessed using a phase contrast microscope. A 50μl each of undiluted, diluted and frozen-thawed semen was mixed with 500μl of 50, 100, 150, 150, 190 or 250 mOsm hypo-osmotic treatments of sodium citrate plus fructose and incubated at 37oC for 1 h. Total number of intact sperm (live) of undiluted, diluted and frozen-thawed were assessed before HOST. Percentage motility decreased (P 0.05), but varied significantly (P<0.05) within bulls. In conclusion, plasma membrane integrity of undiluted and diluted sperm was compromised during freezing and thawing. However, freezing had no effect on acrosomal integrity.