title: Freeze-dried stallion spermatozoa: evaluation of two chelating agents and comparative analysis of three sperm DNA damage assays.
abstract: During the freeze-drying procedure, sperm DNA might become damaged by both freezing and drying stresses. Sperm DNA status can be detected using well-established assays; however, most techniques are expensive and involve elaborate protocols and equipment. Indirect assessments can provide alternative strategies. The objective of this study was to compare a simple test of DNA status using Diff-Quik (DQ) with two established procedures: acridine orange test (AOT) and sperm chromatin dispersion (SCD) on freeze-dried (FD) stallion spermatozoa. Ejaculated spermatozoa from three stallions were freeze-dried in basic medium supplemented with two different chelating agents: EGTA or EDTA. After rehydration, the spermatozoa were subjected to DNA damage detection using a SCDt, AOT and DQ stain simultaneously. The results showed that the DNA damage levels in the EGTA group were significantly lower than those in the EDTA group. AOT detected a significantly higher proportion of spermatozoa with fragmented DNA than DQ and SCD. The results of the SCD test and DQ stain exhibited a significant positive correlation for DNA fragmentation (r = 0.528), whereas a negative correlation was observed between SCD, DQ and AOT (r = -0.134 and r = -0.332 respectively). The present study shows that both the SCD test and DQ assay are effective methods for detecting FD stallion sperm DNA fragmentation, whereas using of AOT is questionable.
authors.affiliation: Reproduction and Obstetric Area, Departamento de Patología Animal, Universidad de Zaragoza, Zaragoza, Spain. maiteor88@gmail.com.
authors.forename: M
authors.initials: M
authors.lastname: Olaciregui
authors.affiliation: Reproduction and Obstetric Area, Departamento de Patología Animal, Universidad de Zaragoza, Zaragoza, Spain.
authors.forename: V
authors.initials: V
authors.lastname: Luño
authors.affiliation: Reproduction and Obstetric Area, Departamento de Patología Animal, Universidad de Zaragoza, Zaragoza, Spain.
authors.forename: J I
authors.initials: JI
authors.lastname: Martí
authors.affiliation: Pharmacology and Physiology Area, Facultad de Veterinaria, Universidad de Zaragoza, Zaragoza, Spain.
authors.forename: J
authors.initials: J
authors.lastname: Aramayona
authors.affiliation: Reproduction and Obstetric Area, Departamento de Patología Animal, Universidad de Zaragoza, Zaragoza, Spain.
authors.forename: L
authors.initials: L
authors.lastname: Gil
mesh_terms: D000165:Acridine Orange; D000818:Animals; D002614:Chelating Agents; D004396:Coloring Agents; D004249:DNA Damage; D004492:Edetic Acid; D004533:Egtazic Acid; D005456:Fluorescent Dyes; D005612:Freeze Drying / Q000379:methods / Q000662:veterinary*; D006736:Horses; D066298:In Vitro Techniques; D008297:Male; D012662:Semen Preservation / Q000009:adverse effects / Q000379:methods / Q000662:veterinary*; D013094:Spermatozoa / Q000378:metabolism*
chemical_list: D002614:Chelating Agents; D004396:Coloring Agents; D005456:Fluorescent Dyes; D004533:Egtazic Acid; D004492:Edetic Acid; D000165:Acridine Orange
keywords: DNA fragmentation; Denaturation; diff-quik; freeze-drying; halomax