title: Equine spermatozoa stored in the epididymis for up to 96h at 4°C can be successfully cryopreserved and maintain their fertilization capacity.
abstract: After injury or death of a valuable male, recovery of epididymal spermatozoa may be the last chance to ensure preservation of its genetic material. The objective of this research was to study the effect of sperm storage, at 4°C up to 96h, in the epididymides obtained from castrated horses and its effect on different functional sperm parameters. Aims were to study the effect of (1) sperm storage on viability and chromatin condensation; (2) pre-incubation of recovered epididymal sperm in the freezing extender, prior cryopreservation, on viability and chromatin condensation; and (3) freezing-thawing on viability, chromatin condensation, ROS generation, protein tyrosine phosphorylation and heterologous fertilization rate (ICSI and IVF using bovine oocytes) of sperm recovered from the epididymis up to 96h post castration. The average volume (720±159μL) and the concentration (6.5±0.4×10(9) spermatozoa/mL) of sperm recovered from the epididymis were not affected by storage. Sperm viability after refrigeration at 4°C for up to72h was similar (P<0.01). The effect of sperm dilution in the freezing media showed similar values up to 48h, while viability was preserved up to 72h (P<0.01). Cryopreserved spermatozoa show similar viability between different storage times. Chromatin condensation was not affected by storage time; however, incubation for 30min in freezing medium and freezing-thawing process induced an increase in the chromatin decondensation. ROS generation was not affected by storage up to 96h. Epididymal storage did not affect sperm protein tyrosine phosphorylation patterns; although the pattern of phosphorylation changed to strong staining of the equatorial segment when the sperm where capacitated in sperm-TALP. Finally, successful and similar pronuclear formation (analyzed by ICSI) and in vitro penetration (evaluated with bovine zone free oocyte) was observed using cryopreserved sperm obtained from prolong epididymal storage at 4°C. In conclusion, cryopreservation of epididymal stallion sperm stored for up to 72h in the epididymis at 4°C, maintain both viability and ability to fertilize in vitro.
authors.affiliation: Department of Physiology, Faculty of Veterinary, University of Murcia, Murcia 30071, Spain.
authors.forename: L A
authors.initials: LA
authors.lastname: Vieira
authors.forename: J
authors.initials: J
authors.lastname: Gadea
authors.forename: F A
authors.initials: FA
authors.lastname: García-Vázquez
authors.forename: K
authors.initials: K
authors.lastname: Avilés-López
authors.forename: C
authors.initials: C
authors.lastname: Matás
mesh_terms: D000818:Animals; D002470:Cell Survival / Q000502:physiology; D015925:Cryopreservation / Q000379:methods / Q000662:veterinary*; D004822:Epididymis / Q000502:physiology*; D006736:Horses; D008297:Male; D013094:Spermatozoa / Q000502:physiology*; D013997:Time Factors; D014021:Tissue Preservation / Q000379:methods / Q000662:veterinary