Changes in Motion Characteristics, Plasma Membrane Integrity, and Acrosome Morphology During Cryopreservation of Buffalo Spermatozoa

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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 triscitric 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 differdue to dilution, cooling, or equilibration (77.3% ± 2.3% and 90.5% ± 1.2%, respectively), but was reduced (P < .05) after freezing and thawing (53.0% ± 4.6% and 48.6% ± 6.5%, respectively). Linear motility of spermatozoa was lower (P < .05) after dilution or equilibration (56.2% ± 2.4%) than after cooling or freezing and thawing (79.6% ± 1.4%). Sperm curvilinear velocity was reduced (P < .05) from 112.4 ± 5.3 μm/sec after dilution to 96.0 ± 5.8 μm/s after cooling, and from 87.6 ± 4.1 μm/s after equilibration to 69.4 ± 2.0 μm/s after freezing and thawing. Sperm lateral head displacement differed (P < .05) after each stage (ie, dilution, 3.9 ± 0.2 μm; cooling, 2.3 ± 0.2 μm; equilibration, 3.1 ± 0.3 μm; and freezing and thawing, 1.7 ± 0.2 μm). Spermatozoa with intact plasma membranes were 80.2% ± 3.9% after dilution, reduced (P < .05) to 60.4% ± 5.6% after equilibration, and then to 32.6% ± 3.8% after freezing and thawing. The percentage of spermatozoa with normal acrosomes remained higher after dilution, cooling, or equilibration (73.2% ± 2.4%) than after freezing and thawing (61.8% ± 2.4%; P < .05). In conclusion, the maximal damage to the motility apparatus, plasma membrane, and acrosomal cap of buffalo spermatozoa occurs during freezing and thawing followed by equilibration.

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