Changes in motion characteristics, plasma membrane integrity, and acrosome morphology during cryopreservation of buffalo spermatozoa

Abstract

Motion characteristics, plasma membrane integrity, and acrosome morphology of buffalo spermatozoa after different stages of cryopreservation (ie, dilution, cooling to 4°C, equilibration at 4°C, and freezing and thawing) were examined. Semen ejaculates from 4 buffalo bulls were pooled (n = 5), diluted in tris-citric acid extender, cooled to 4°C over 2 hours, equilibrated at 4°C for 4 hours, dispensed into 0.5-mL straws, and frozen in a programmable cell freezer before plunging into liquid nitrogen. Frozen semen was thawed at 37°C for 15 seconds. After completion of each stage, sperm motion characteristics, plasma membrane integrity, and acrosomal morphology were determined using computer-assisted semen analysis, hypo-osmotic swelling assay, and phase-contrast microscopy, respectively. Data were presented as mean ± standard error of the mean. Visual and computerized motility did not differ due to dilution, cooling, or equilibration (77.3% ± 2.3% and 90.5% ± 1.2%, respectively), but was reduced (P