Muhammed Irfan-Rehman Khan

Effect Of Osmotic Pressure On The Membrane Integrity Of Frozen-Thawed Buffalo Bull Spermatozoa - 2005

In the first experiment, semen from seven Nili-Ravi buffalo bulls was used to study plasma membrane integrity of freshly collected (raw) and frozen-thawed sperm using the hypo-osmotic swelling test (HOST). For this purpose, percentage motility, integrity of plasma membrane and acrosome was assessed by a phase contrast microscope, HOST plus eosin-nigrosin staining and normal apical ridge test. respectively. 50fJI each of raw and frozen-thawed semen was mixed with 500fJI of 50, 100, 150, 190 or 250 mOsm hypo-osmotic treatments of sodium citrate plus fructose and incubated at 37°C for 1 h. Integrity of sperm plasma membrane was assessed before and after hypo-osmotic treatments to estimate the extent of damage for each hypo-osmotic treatment. In raw semen, the number of swollen sperm was higher (P<0.05) at 50, 100, 150 and 190 mOsm as compared to 250 mOsm. A positive but non-significant correlation (P>0.05) was found between percentage of swollen and live sperm at 100, 150, 190 mOsm. Similarly, a positive but non-significant correlation (P>0.05) was found between percentage of swollen sperm and motility at 50, 100, 150, 190 and 250 mOsm. In frozen-thawed semen, the number of swollen sperm w.as higher (P<0.05) at 50 and 100 mOsm as compared to 150, 190 and 250 mOsm and this number decreased significantly (P<0.05) and gradually from 82.6±5.99 at 150 mOsm to 69.7±5.49 at 190 mOsm and 42.6±4.07 at 250 mOsm. A positive but non-significant correlation (P>0.05) was found between percentage of swollen and live sperm and between percentage of swollen sperm and motility at 50, 100 150, 190 and 250 mOsm. The number of sperm with intact acrosome did not differ (P>0.05) in raw and frozen-thawed semen among treatments. Percentag~ motility in raw semen was highe~' (81%) as compared to frozen-thawed semen (60%). Damage to plasma membrane'was higher (P<0.05) at 50 mOsm (59% raw vs. 70% frozen-thawed) as, compared to other hypo-osmotic treatments, while minimum damage occurred at 250 mOsm (4.1% raw vs. 9.7% frozen-thawed). In the second experiment, hypoosmotic swelling test (HOST) plus eosin-,nigrosin staining and normal apical ridge test (NAR) were used to determine integrity of plasma membrane and acrosome of raw, diluted (cooled to 5°C) and frozen-thawed sperm. Semen from seven bulls was used in this study. For diluted and frozen-thawed sperm, three straws were pooled at 37°C. Percentage motility of raw, diluted and frozen-thawed sperm was assessed using a phase contrast microscope. 501-11 each of raw, diluted and frozen-thawed sperm was mixed with 5001-11 of 50, 100, 150, 150, 190 or 250 mOsm hypo-osmotic treatments of sodium citrate plus fructose and incubated at 37°C for 1 h. Total number of intact (live) sperm of raw, diluted and frozen-thawed semen was assessed before HOST. Diluted sperm showed higher (P<0.05) swellings of plasma membrane at 50
and 100 mOsm than raw sperm. Similarly, swellings of diluted sperm were' higher (P<0.05) than frozen-thawed sperm. Swellings of raw sperm were significantly higher (P<0.05) at 100, 150, 190 and 250 mOsm than frozenthawed sperm. A significant decrease (P<0.05) was found among percentage motility of raw (81±1.57), diluted (69.6±2.24) and frozen thawed (60.1 ±1.34) sperm. Live sperm were higher (P<0.05) in raw (174.4±7.33) and diluted semen (175.6±3.76) as compared to frozen-thawed semen (142.3±4.84). Although integrity of the acrosome of raw, diluted and frozen-thawed sperm did not differ (P>0.05), significan~ variation was found within bulls. In conclusion, fresh and frozen-thawed sperm behaved differently to HOST and the number of swollen sperm was higher in raw as compared to frozenthawed semen. Plasma membrane' integrity of raw and diluted sperm was compromised during freezing and thawing. However, freezing had no effect on acrosome integrity. Moreover, 100, 150 and 190. mOsm were found suitable to perform HOST.



Department of Theriogenology

0911,T


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