Abstract
A major advantage of intracytoplasmic sperm injection (ICSI) is the ability to obtain several in vitro produced embryos from a single straw of frozen semen. Thawing and refreezing of the semen at a low concentration would result in large number of straws that could be reselled. However, equine spermatozoa are
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very susceptible to cold-shock and earlier results indicated that sperm quality declines remarkably during refreezing. Stabilization of the plasma membrane by cholesterol during processing may improve the resistance of sperm to repeated freeze-thaw cycles. In the current study, the effect of treating equine spermatozoa with cholesterol-loaded cyclodextrin (CLC) prior to the first freezing, on sperm quality after thawing and subsequent refreezing and thawing, was evaluated. Ejaculates (n=3) were split and treated with CLC (0.5, 1.5, 3.0, & 4.5 mg/mL) or without (negative control). Staining with merocyanine and YoPro-1 indicated that CLC treatment decreased membrane fluidity in live cells in a dose-dependent manner by up to 30%. However, fluidity of the control samples decreased to the same extent once samples were exposed to an egg yolk containing freezing extender. Total motility after thawing was improved for samples treated with 3.5 mg/mL CLC (41.1 ± 21.9%) when compared to controls (29.9 ± 16.0%). The percentage of live, acrosome intact spermatozoa (propidium iodide, PNA-FITC & PSA-FITC negative) was not affected by CLC treatment. Refreezing of the semen resulted in an almost complete loss of motile cells irrespective of the CLC treatment. A second, preliminary experiment focusing on processing speed and dilution rate for refreezing of the control samples (n=2) indicated that quick processing and moderate dilution result in higher total motility (17.9 ± 11.3%) when compared to slower processed samples at high dilution rates (1.4 ± 1.9%).
In conclusion, the results of this study demonstrate that CLC treatment is able to decrease the fluidity of the plasma membrane of live sperm cells that haven’t been exposed to semen extender and improve sperm motility after one cycle of freezing and thawing. Nonetheless, CLC treatment prior to the first freezing does not improve sperm quality after two cycles of freezing and thawing. An optimized quick processing of sperm after thawing without CLC treatment may offer a perspective for future refreezing of stallion sperm for ICSI.
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