Comparison of two methods for obtaining spermatozoa from the cauda epididymis of Iberian red deer

We have compared two methods for salvaging epididymal sperm from post-mortem samples from Iberian red deer. Of each pair of testicles (29 samples), one cauda epididymis was processed by means of cuts (sperm was immediately diluted with extender) and the other was detached from the corpus and flushed from the vas deferens with 1 mL of extender. Sperm was processed for cryopreservation, and analyzed just after recovery, pre-freezing and post-thawing. Total spermatozoa recovered, contamination (concentration of epididymal cells and red blood cells (RBCs)) and quality (motility by CASA, and acrosomal status, viability and mitochondrial status by flow cytometry) were used to compare both methods. The number of recovered spermatozoa was similar for both methods. Contamination was higher for the cuts method, but when considering the final dilution before freezing, only RBCs concentration was significantly higher. Motility was similar just after extraction, but higher for both pre-frozen and post-thawed flushed sperm. Pre-freezing acrosomal status (P < 0.05) and viability (P < 0.1) were better for flushing; however post-thawing results were similar for the two methods. A clustering analysis using CASA data showed that the subpopulation pattern of motile sperm was different depending on the method, being better for flushing. With regard to yield, lower contamination (especially RBCs) and, in general, better quality results, flushing seems to be a more recommendable method for post-mortem sperm recovery. The cuts method may be more practical on certain occasions, but care must be taken in order to achieve rapid extension of the sample and to avoid contamination in order to improve sample condition.