title: Freeze-dried dog sperm: Dynamics of DNA integrity.
abstract: Freeze-drying (FD) has been proposed as an alternative method to preserve spermatozoa. During the FD procedure, sperm DNA might become damaged by both freezing and drying stresses caused by the endonucleases, the oxidative stress and the storage conditions. We examined the DNA integrity of dog sperm freeze-dried with two kinds of chelating agents in FD buffers and storage at two different temperatures. Ejaculated sperm from four dogs were suspended in basic medium (10 mM Tris-HCl buffer+50 mM NaCl) supplemented with 50 mM EGTA or with 50 mM EDTA and then freeze-dried. Sperm samples were stored at 4°C as room temperature, and the analysis of DNA damage was performed after a month and 5 months of storage using a Sperm Chromatin Dispersion test. We found four different sperm populations according to the size of the halos around the sperm head: (1) absent halo, (2) <6 μm, (3) 6-10 μm, (4) >10 μm. All of them coexisted in each freeze-dried dog semen samples and differed significantly among different treatments. The highest percentage of spermatozoa with halo >10 μm was obtained when the semen samples were freeze-dried in EDTA medium and stored at room temperature for five months. Results suggested that both, the kind of chelating agent as well as storage temperature and period, influenced DNA integrity of freeze-dried dog sperm.
authors.affiliation: Reproduction and Obstetric Area, Departamento de Patología Animal, Facultad de Veterinaria, Universidad de Zaragoza, Miguel Servet 177, 50013 Zaragoza, Spain. Electronic address: maiteor88@gmail.com.
authors.forename: M
authors.initials: M
authors.lastname: Olaciregui
authors.affiliation: Reproduction and Obstetric Area, Departamento de Patología Animal, Facultad de Veterinaria, Universidad de Zaragoza, Miguel Servet 177, 50013 Zaragoza, Spain.
authors.forename: V
authors.initials: V
authors.lastname: Luño
authors.affiliation: Reproduction and Obstetric Area, Departamento de Patología Animal, Facultad de Veterinaria, Universidad de Zaragoza, Miguel Servet 177, 50013 Zaragoza, Spain.
authors.forename: N
authors.initials: N
authors.lastname: Gonzalez
authors.affiliation: Infectious Diseases Area, Departamento de Patología Animal, Facultad de Veterinaria, Universidad de Zaragoza, Miguel Servet 177, 50013 Zaragoza, Spain.
authors.forename: I
authors.initials: I
authors.lastname: De Blas
authors.affiliation: Reproduction and Obstetric Area, Departamento de Patología Animal, Facultad de Veterinaria, Universidad de Zaragoza, Miguel Servet 177, 50013 Zaragoza, Spain.
authors.forename: L
authors.initials: L
authors.lastname: Gil
mesh_terms: D000818:Animals; D002614:Chelating Agents / Q000494:pharmacology*; D003451:Cryoprotective Agents / Q000494:pharmacology; D004247:DNA / Q000235:genetics*; D004249:DNA Damage / Q000235:genetics; D004285:Dogs; D005612:Freeze Drying / Q000379:methods*; D008297:Male; D012662:Semen Preservation / Q000009:adverse effects / Q000379:methods / Q000662:veterinary*; D013077:Sperm Head / Q000502:physiology*; D020554:Sperm Injections, Intracytoplasmic / Q000662:veterinary; D013696:Temperature; D014325:Tromethamine
chemical_list: D002614:Chelating Agents; D003451:Cryoprotective Agents; D014325:Tromethamine; D004247:DNA
keywords: DNA fragmentation; Dog sperm; Freeze-drying; Halomax