title: Assessment of the efficacy of Sephadex G-15 filtration of bovine spermatozoa for cryopreservation.
abstract: Semen from five dairy AI bulls was split-filtered through a Sephadex G-15 filter and frozen in a Tris-citric acid buffer egg yolk-based extender. The effect of filtration was studied morphologically for individual sperm abnormalities. Computer-assisted sperm analysis (CASA) was used for motility and sperm motion assessment. Flow cytometry was used to disclose sperm viability (SYBR-14/PI), mitochondrial membrane potential (Mitotracker Deep Red/SYBR 14), acrosome integrity (SYBR 14/PE-PNA/PI), plasma membrane stability (Merocyanine 540/YO-PRO 1/Hoechst 333342), and chromatin stability (acridine orange staining). Filtration significantly reduced the concentration of recovered spermatozoa (P < 0.01), but improved semen quality, reducing the number of spermatozoa with various forms of morphological defects. Filtration also affected percentages of sperm motility after equilibration and after freezing/thawing. Sperm motion characteristics were, however, not significantly affected by filtration at any stage of the cryopreservation protocol, including post-extension, equilibration, or freezing/thawing. Filtration enhanced sperm viability after thawing (P < 0.05), but had no significant effect (P > 0.05) on recovery of spermatozoa with high mitochondrial potential, intact acrosomes, or preserved sperm chromatin structure. Sperm plasma membrane stability was also not affected by the filtration method used (P > 0.05). It can be concluded that filtration effectively separates weaken or abnormal spermatozoa in pre-freezing semen samples and therefore the procedure could be recommended to improve post-thaw sperm viability of selected, fertile sires.
authors.affiliation: Department of Obstetrics and Gynaecology, Lithuanian Veterinary Academy, Lithuania.
authors.forename: Aloyzas
authors.initials: A
authors.lastname: Januskauskas
authors.forename: Kristina
authors.initials: K
authors.lastname: Lukoseviciute
authors.forename: Szabolcs
authors.initials: S
authors.lastname: Nagy
authors.forename: Anders
authors.initials: A
authors.lastname: Johannisson
authors.forename: Heriberto
authors.initials: H
authors.lastname: Rodriguez-Martinez
mesh_terms: D000177:Acrosome / Q000648:ultrastructure; D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000502:physiology / Q000648:ultrastructure; D002469:Cell Separation / Q000379:methods / Q000662:veterinary; D002470:Cell Survival; D002843:Chromatin / Q000648:ultrastructure; D002850:Chromatography, Gel / Q000379:methods / Q000662:veterinary*; D015925:Cryopreservation / Q000662:veterinary*; D003911:Dextrans; D005434:Flow Cytometry; D007202:Indicators and Reagents; D008297:Male; D008564:Membrane Potentials; D008928:Mitochondria / Q000648:ultrastructure; D013076:Sperm Count; D013081:Sperm Motility; D013094:Spermatozoa / Q000002:abnormalities / Q000648:ultrastructure
chemical_list: D002843:Chromatin; D003911:Dextrans; D007202:Indicators and Reagents; C025614:sephadex