C-1012
2014
Salmon, V. | Leclerc, P. | Bailey, J.
The success of semen cryopreservation mainly resides in maintaining sperm membrane integrity and function after thawing. Cholesterol-loaded cyclodextrin (CLC) is used for in vitro incorporation of cholesterol into cells to protect them against cold temperature. We hypothesized that CLC treatment enhances sperm cholesterol content to increase its tolerance to osmotic shock and cryo-resistance, thereby improving fertility. Alpine goat sperm were treated ±3mg/ml CLC and lipids extracted by the Folch method, then cholesterol was quantified with Liebermann-Burchard reagent, which confirmed that CLC treatment increases the cholesterol content of fresh sperm in either PBS or skim milk extender. Sperm were then treated±CLC in skim milk extender and cryopreserved. After thawing, sperm cholesterol dramatically fell, even in the presence of CLC, and likely explains the mechanism of “cryo-capacitation”. However, CLC treatment helped maintain a normal pre-freeze cholesterol level in the sperm after cryopreservation. Labelling sperm cholesterol with filipin confirmed this result on sperm membranes. Furthermore, fresh sperm in skim milk extender treated 3mg/ml CLC and incubated for 20min in hypo-, iso- and hyperosmotic PBS have increased hypoosmotic tolerance, corresponding to the beneficial effects of CLC at thawing. As expected, CLC treatment in skim milk extender improved sperm viability, motility and acrosome integrity after thawing. Furthermore, ultrasonography after artificial insemination revealed that sperm treated with CLC are 10% more fertile than control semen without CLC.
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