title: Influence of cooling and thawing conditions and cryoprotectant concentration on frozen-thawed survival of white-naped crane (Antigone vipio) spermatozoa.
abstract: To assist in genetic resource management and recovery efforts of the white-naped crane (Antigone vipio), we conducted two experiments to evaluate the effect of cooling condition, thawing rate, and cryoprotectant concentration on sperm survival post-thaw. Semen was collected from four mature males during breeding season (March and April) and evaluated for volume, sperm concentration, motility, and membrane integrity. In Experiment 1, ejaculates (n = 8) were diluted with Beltsville Poultry Semen Extender (BPSE) containing 10% dimethylsulfoxide (Me2SO) and frozen using either one (average cooling rate = 2.5 °C/min) or two step (average cooling rate = 7 and 9 °C/min, respectively) cooling method. The frozen samples were thawed using one of two thawing rates: 37 °C 30 s vs. 4 °C 1 min. In Experiment 2, samples were diluted with crane semen extender containing either 6% or 10% Me2SO, frozen using two-step method and then thawed at 37 °C for 30 s. Both cooling condition (two-step > one-step) and thawing rate (37 °C 30 s > 4 °C 1 min) impacted sperm motility, progression and kinetic characteristics (P < 0.05), but did not (P > 0.05) affect plasma membrane or acrosomal integrity. Concentration of Me2SO did not impact frozen-thaw survival. We conclude that white-naped crane sperm cryopreserved using a combination of two-step cooling and thawing at 37 °C 30 s was superior to other cooling and thawing combinations regarding to sustaining sperm motility with good motility kinetics. Findings represent the first steps towards the development of effective cryopreservation protocols and establishment of a genome resource bank for this threatened species.
authors.affiliation: Center for Species Survival, Smithsonian Conservation Biology Institute, National Zoological Park, 1500 Remount Road, Front Royal, VA 22630, USA; Department of Obstetrics, Gynaecology and Reproduction, Faculty of Veterinary Science, Chulalongkorn University, Bangkok 10330, Thailand.
authors.forename: Saritvich
authors.initials: S
authors.lastname: Panyaboriban
authors.affiliation: Center for Species Survival, Smithsonian Conservation Biology Institute, National Zoological Park, 1500 Remount Road, Front Royal, VA 22630, USA.
authors.forename: Budhan
authors.initials: B
authors.lastname: Pukazhenthi
authors.affiliation: Center for Species Survival, Smithsonian Conservation Biology Institute, National Zoological Park, 1500 Remount Road, Front Royal, VA 22630, USA; Department of Animal and Avian Sciences, 1413 Animal Sciences Center, University of Maryland, College Park, MD 20742, USA.
authors.forename: Megan E
authors.initials: ME
authors.lastname: Brown
authors.affiliation: Center for Species Survival, Smithsonian Conservation Biology Institute, National Zoological Park, 1500 Remount Road, Front Royal, VA 22630, USA.
authors.forename: Chris
authors.initials: C
authors.lastname: Crowe
authors.affiliation: Center for Species Survival, Smithsonian Conservation Biology Institute, National Zoological Park, 1500 Remount Road, Front Royal, VA 22630, USA.
authors.forename: Warren
authors.initials: W
authors.lastname: Lynch
authors.affiliation: Center for Species Survival, Smithsonian Conservation Biology Institute, National Zoological Park, 1500 Remount Road, Front Royal, VA 22630, USA; Sálim Ali Centre for Ornithology and Natural History, Anaikatty, Coimbatore 641 108, India.
authors.forename: Ram P
authors.initials: RP
authors.lastname: Singh
authors.affiliation: Department of Obstetrics, Gynaecology and Reproduction, Faculty of Veterinary Science, Chulalongkorn University, Bangkok 10330, Thailand.
authors.forename: Mongkol
authors.initials: M
authors.lastname: Techakumphu
authors.affiliation: Center for Species Survival, Smithsonian Conservation Biology Institute, National Zoological Park, 1500 Remount Road, Front Royal, VA 22630, USA. Electronic address: songsasenn@si.edu.
authors.forename: Nucharin
authors.initials: N
authors.lastname: Songsasen
mesh_terms: D000818:Animals; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology*; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D008297:Male; D012662:Semen Preservation / Q000379:methods*; D013081:Sperm Motility / Q000187:drug effects; D013094:Spermatozoa / Q000187:drug effects
chemical_list: D003451:Cryoprotective Agents; D004121:Dimethyl Sulfoxide
keywords: Cooling condition; Cryopreservation; Me(2)SO; Motility; Sperm; Thawing rate; White naped crane