Comparison of Centrifugation and Noncentrifugation Methods to Cryopreserve Stallion Epididymal Semen

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Abstract

Most epididymal flushes are performed after a catastrophic injury, acute, or chronic illness and are the last chance to save genetic material from a valuable stallion; this not only prevents test freezing for the particular animal but also means that the starting semen quality is likely poor. This necessitates that the protocol used for epididymal flushes be optimized to achieve the best possible recovery of the least damaged sperm. The objective of this study was to compare the effects of direct flushing of the epididymis with freezing extender to flushing with cooling extender and centrifugation. Epididymides were dissected away from the testes, and each vas deferens was catheterized. For the centrifugation method, the epididymis was flushed using 5 mL of a skim milk–based extender, followed by 20 mL of air, and then, semen was diluted to 50 million/mL before centrifugation and processing. Epididymides used for the noncentrifugation method were flushed directly with a commercial semen freezing extender. Semen was loaded in 0.5-mL straws at 200 million/mL, cooled and frozen. Post-thaw samples were evaluated for total and progressive sperm motility using a computer-automated sperm analyzer. Additionally, post-thaw samples were stained with fluorescent probes and subjected to flow cytometry for assessment of sperm viability, mitochondrial potential, acrosome integrity, and chromatin stability. No significant differences were observed between the two groups for any of the sperm parameters evaluated. In conclusion, although centrifugation does not appear to be detrimental to epididymal sperm, it also does not seem necessary for semen processing.

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