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

Abstract

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.

Viability of ovine spermatozoa collected from epididymides stored at 18°-25°C for 48 hours post mortem

“…Based on our results we hypotheses that ovine epididymal spermatozoa exposed to ambient temperature could yield good fertility up to 24 hours post mortem, however efforts should be focused on preservation (refrigeration or cryopreservation) of epididymal spermatozoa as soon as possible. -Pastor et al (2006) compared a slicing method with retrograde flushing for recovery of red deer spermatozoa, these authors found no difference between the methods in sperm recovery and motility immediately after collection but identified better motility, acrosome integrity and viability in sperm collected by retrograde flushing when spermatozoa were diluted and cooled for two hours. In our preliminary work, we identified limitations of the retrograde flushing method on ram cauda epididymis.…”

“…Petri plates were angled so that the extender washed the spermatozoa to accumulate at one end of the plate. After five minutes a pipette was used to collect the diluted spermatozoa from the Petri dish and the sample was placed in a conical tube (Falcon BD) (Martinez-Pastor et al, 2006).…”

Conservation of endangered animals: From biotechnologies to digital preservation

“…Nowadays concrete examples of application of post-mortem sperm recovery from the epididymis are available for conservation of endangered species [37] and in constructing semen banks [38][39][40]. Preservation of epididymal sperm from accidental dead or slaughtered animals has been reported in several domestic and wild species as boar [41], cattle [42,43], goat [44], ibex [45,46], red deer [47] and sheep [38,48]. However epididymal sperm extraction sometimes is not easily practicable in marginal areas, due to the lack of facilities and expertise near the farming areas of local breeds.…”

Fertility of undiluted ram epididymal spermatozoa stored for several days at 4°C

“…Sperm collected from caudae epididymides has been used in reproductive techniques developed for several species, including the mouse , dog (Yu and Leibo, 2002;Garcia-Macias et al, 2006a), deer (Hishinuma et al, 2003;Martinez-Pastor et al, 2006), pig (Kikuchi et al, 1998), ram (Kaabi et al, 2003), bull (Foote, 2000), horse (Vieira et al, 2012), bison (Krishnakumar et al, 2011), bear (Garcia-Macias et al, 2006b) and even humans (McLachlan, 1998). The sampling of epididymal spermatozoa has been reported after mincing or cutting the epididymal tubule and collecting the exudate directly (Martins et al, 2009;Tamayo-Canul et al, 2011b) or diluting in a medium solution (Hori et al, 2005b;Ehling et al, 2006).…”

“…The sampling of epididymal spermatozoa has been reported after mincing or cutting the epididymal tubule and collecting the exudate directly (Martins et al, 2009;Tamayo-Canul et al, 2011b) or diluting in a medium solution (Hori et al, 2005b;Ehling et al, 2006). To avoid any blood contamination and to obtain a maximum number of spermatozoa, epididymal sampling has also been performed by retroflushing the epididymal lumen content from the deferent duct with air (Kikuchi et al, 1998;Vieira et al, 2012), with physiological solution (Dacheux, 1980;Martinez-Pastor et al, 2006), sodium citrate-egg yolk or paraffin oil (Dacheux et al, 2006). Although some differences have been reported between caudal and ejaculated sperm (GarciaMacias et al, 2006a), the fertility of epididymal sperm used either fresh or frozen-thawed for insemination has been found to be similar to ejaculated samples (Ehling et al, 2006;Varisli et al, 2009;Alvarez et al, 2012).…”