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Effect of cholesterol on post-thaw motility and plasma membrane damage of equine spermatozoa
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This study was designed to investigate the cryoprotective effects of cholesterol incorporation into plasma membranes of equine spermatozoa using a novel method to alter membrane cholesterol content with a cholesterol-methyl-p-cyclodextrin complex. Experiment 1 was conducted to evaluate effects of four cholesterol concentrations (0.0, 0. 125, 0.25 or 0. 5 mM) five incubation times (0, 15, 30, 45 or 60 minutes), and two incubation temperatures (24 or 37'C) on post-thaw computerized motility parameters. Two centrifugation media were used throughout the study: a non-fat skim milk glucose-sucrose extender (MK) and a modified Tyrode's medium (TALP). The MK media generally yielded better spermatozoal motility characteristics than TALP media. Cholesterol treatments tended to improve post-thaw motility over control media, with significant improvement in percent total motility with 0. 125 mM cholesterol (p < 0. 05). Cholesterol incorporation did not affect velocity parameters (p>0.05). Increasing incubation time tended to decrease percent spermatozoal motility. Incubation for 15 minutes tended to improve motility and preserved velocity parameters over longer incubation times. An incubation temperature of 24'C also tended to increase motility parameters when compared to 37'C. In Experiment 2, tritium-labeled cholesterol was utilized to verify spermatozoal incorporation of cholesterol in ejaculates of three stallions. Four different concentrations of tritiated cholesterol (0. 0, 0. 125, 0.25 or 0. 50 mM) in MK or TALP media were used to evaluate effect of concentration on membrane uptake. There was significant (p<0.05) incorporation of cholesterol at all concentrations in both extenders when compared to the control. In Experiment 3, cryoprotective effects of cholesterol on spermatozoal plasma membranes were evaluated. Ejaculates from six stallions were cryopreserved to evaluate effect of centrifugation extender (MK or TALP) and added cholesterol (0. 125 mM) on post-thaw spermatozoal motility parameters and plasma membrane integrity. To evaluate membrane damage, spermatozoa were stained with carboxyfluorescein diacetate and propidium iodide fluorescent stains. Addition of cholesterol to either extender significantly improved (p < 0.05) percentages of total, progressive and rapid spermatozoal motility in post-thaw samples. Cholesterol incorporation also significantly decreased the number of spermatozoa with post-thaw damage to the plasma membrane in MK (p<0.05) and TALP (p<0.06) media.
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Combes, Gayla Beth (1997). Effect of cholesterol on post-thaw motility and plasma membrane damage of equine spermatozoa. Master's thesis, Texas A&M University. Available electronically from
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