title: Spermatozoa from the maned wolf (Chrysocyon brachyurus) display typical canid hyper-sensitivity to osmotic and freezing-induced injury, but respond favorably to dimethyl sulfoxide.
abstract: We assessed the influences of medium osmolality, cryoprotectant and cooling and warming rate on maned wolf (Chrysocyon brachyurus) spermatozoa. Ejaculates were exposed to Ham's F10 medium (isotonic control) or to this medium plus NaCl (350-1000mOsm), sucrose (369 and 479mOsm), 1M glycerol (1086mOsm) or dimethyl sulfoxide (Me2SO, 1151mOsm) for 10 min. Each sample then was diluted back into Ham's medium and assessed for sperm motility and plasma membrane integrity. Although glycerol and Me2SO had no influence (P>0.05), NaCl and sucrose solutions affected sperm motility (P<0.05), but not membrane integrity. Motility of sperm exposed to <600mOsm NaCl or sucrose was less (P<0.05) than fresh ejaculate, but comparable (P>0.05) to the control. As osmolality of the NaCl solution increased, motility decreased to <5%. In a separate study, ejaculates were diluted in Test Yolk Buffer containing 1M glycerol or Me2SO and cooled from 5°C to -120°C at -57.8°C, -124.2°C or -67.0°C/min, frozen in LN2, thawed in a water bath for 30s at 37°C or 10s at 50°C, and then assessed for motility, plasma- and acrosomal membrane integrity. Cryopreservation markedly (P<0.05) reduced sperm motility by 70% compared to fresh samples. Higher (P<0.05) post-thaw motility (20.0±1.9% versus 13.5±2.1%) and membrane integrity (51.2±1.7% versus 41.5±2.2%) were observed in samples cryopreserved in Me2SO than in glycerol. Cooling rates influenced survival of sperm cryopreserved in glycerol with -57.8°C/min being advantageous (P<0.05). The findings demonstrate that although maned wolf spermatozoa are similar to domestic dog sperm in their sensitivity to osmotic-induced motility damage, the plasma membranes tolerate dehydration, and the cells respond favorably to Me2SO as a cryoprotectant.
authors.affiliation: Center for Species Survival, Smithsonian Conservation Biology Institute, National Zoological Park, Front Royal, VA, USA; Department of Environmental Science and Policy, George Mason University, Fairfax, VA, USA.
authors.forename: Amy E M
authors.initials: AE
authors.lastname: Johnson
authors.affiliation: New Century College, George Mason University, Fairfax, VA, USA.
authors.forename: Elizabeth W
authors.initials: EW
authors.lastname: Freeman
authors.affiliation: Center for Species Survival, Smithsonian Conservation Biology Institute, National Zoological Park, Front Royal, VA, USA.
authors.forename: David E
authors.initials: DE
authors.lastname: Wildt
authors.affiliation: Center for Species Survival, Smithsonian Conservation Biology Institute, National Zoological Park, Front Royal, VA, USA. Electronic address: songsasenn@si.edu.
authors.forename: Nucharin
authors.initials: N
authors.lastname: Songsasen
mesh_terms: D000818:Animals; D046028:Canidae / Q000502:physiology; D015925:Cryopreservation / Q000379:methods / Q000662:veterinary*; D003451:Cryoprotective Agents / Q000378:metabolism*; D004121:Dimethyl Sulfoxide / Q000378:metabolism*; D004285:Dogs; D005990:Glycerol / Q000378:metabolism; D008297:Male; D009997:Osmotic Pressure; D012662:Semen Preservation / Q000379:methods / Q000662:veterinary*; D013081:Sperm Motility; D013094:Spermatozoa / Q000166:cytology* / Q000378:metabolism
chemical_list: D003451:Cryoprotective Agents; D005990:Glycerol; D004121:Dimethyl Sulfoxide
keywords: Cooling; Dimethyl sulfoxide; Glycerol; Maned wolf; Osmotic stress; Sperm cryopreservation