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

Abstract

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) among undiluted (81.0 ± 1.57), diluted (69.6 ± 2.24) and frozen thawed (60.1 ± 1.34) sperm. Swellings of plasma membrane of diluted sperm were higher (P<0.05) at 50 and 100 mOsm than undiluted sperm. Similarly, swellings of diluted sperm were higher (P<0.05) than frozen-thawed sperm. Swellings of undiluted sperm were higher (P<0.05) at 100, 150, 190 and 250 mOsm than frozenthawed sperm. Live sperm were higher (P<0.05) in undiluted (174.4 ± 7.33) and diluted (175.6 ± 3.76) as compared to frozen-thawed (142.3 ± 4.84) semen. Integrity of acrosome in undiluted, diluted and frozen-thawed sperm did not differ (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.