title: Effect of different disaccharides on the integrity and fertilising ability of freeze-dried boar spermatozoa: a preliminary study.
abstract: Freeze-drying spermatozoa is a developing technique that facilitates semen storage and transport. However, freeze-dried sperm exhibits impaired DNA integrity, which is associated with reduced fertilizing ability. Boar spermatozoa were freeze-dried in three different freeze-drying EDTA buffers with trehalose (75mM) and lactose (75mM) (EDTA-TL), (2) with sucrose (75mM) and lactose (75mM) (EDTA-SL) or just lactose (150mM) (EDTA-LL) using two freeze-drying protocols. In experiment 1 a one-step protocol was used and in experiment 2 a two-steps protocol was used. Spermatozoa were stored in1.5 mL cryo-tubes and 1.5 mL glass ampules at both 16 degree C and 25 degree C for 1 month. Successfully freeze-dried spermatozoa were stained with acridine-orange to assess chromatin stability. Freeze-drying was most successful when the 2-step protocol was used (experiment 2). Chromatin stability was greater in samples stored in glass ampules compared to cryo tubes. Chromatin stability was also greater in samples freeze-dried in EDTA-LL compared to EDTA-SL or EDTA-TL buffers. Spermatozoa freeze-dried in EDTA-LL and stored for 14 and 28 days at either 16 degree C or 25 degree C were utilized for ICSI. Two pronuclear formation wasgreatest using spermatozoa stored at 25 degree C (69.23%) and for 28 days (50%). Although 16 degree C spermatozoa samples had better stable chromatin, 25 degree C spermatozoa samples offered better two pronuclear formation results. In conclusion, boar spermatozoa freeze-dried using media containing disaccharides exhibit high chromatin stability and are able to fertilise oocytes following ICSI. Disaccharides may therefore advance the development of freeze-drying techniques for spermatozoa enabling ease of sperm storage and transportation.
authors.affiliation: Department of Animal Pathology, Obstetrics and Reproduction Area, Faculty of Veterinary Medicine, Universidad de Zaragoza, Zaragoza, Spain.
authors.forename: A
authors.initials: A
authors.lastname: Garcia
authors.affiliation: Department of Animal Pathology, Obstetrics and Reproduction Area, Faculty of Veterinary Medicine, Universidad de Zaragoza, Zaragoza, Spain.
authors.forename: L
authors.initials: L
authors.lastname: Gil
authors.affiliation: Department of Animal Pathology, Obstetrics and Reproduction Area, Faculty of Veterinary Medicine, Universidad de Zaragoza, Zaragoza, Spain. claramalo@hotmail.com.
authors.forename: C
authors.initials: C
authors.lastname: Malo
authors.affiliation: Department of Animal Pathology, Obstetrics and Reproduction Area, Faculty of Veterinary Medicine, Universidad de Zaragoza, Zaragoza, Spain.
authors.forename: F
authors.initials: F
authors.lastname: Martinez
authors.affiliation: Department of Animal Production, Welfare and Veterinary Sciences, Harper Adams University, Newport, Shropshire, UK.
authors.forename: C
authors.initials: C
authors.lastname: Kershaw-Young
authors.affiliation: Department of Animal Pathology, Infectious Disease Area, Faculty of Veterinary Medicine, Universidad de Zaragoza, Zaragoza, Spain.
authors.forename: I
authors.initials: I
authors.lastname: de Blas
mesh_terms: D000165:Acridine Orange; D000818:Animals; D002843:Chromatin / Q000737:chemistry / Q000187:drug effects*; D005307:Fertilization in Vitro; D005612:Freeze Drying; D007785:Lactose / Q000494:pharmacology; D008297:Male; D009865:Oocytes / Q000166:cytology / Q000254:growth & development; D020011:Protective Agents / Q000494:pharmacology*; D012662:Semen Preservation; D013081:Sperm Motility / Q000187:drug effects; D013094:Spermatozoa / Q000166:cytology / Q000187:drug effects* / Q000502:physiology; D013395:Sucrose / Q000494:pharmacology; D013552:Swine; D014199:Trehalose / Q000494:pharmacology
chemical_list: D002843:Chromatin; D020011:Protective Agents; D013395:Sucrose; D014199:Trehalose; D000165:Acridine Orange; D007785:Lactose