title: An investigation into the similarities and differences governing the cryopreservation success of koala (Phascolarctos cinereus: goldfuss) and common wombat (Vombatus ursinus: shaw) spermatozoa.
abstract: The aim of this study was to determine the relative cryopreservation success of koala and wombat spermatozoa and to investigate reasons for their respective post-thaw survival by examining the sperm's response to a range of osmotic media and determining the presence and distribution of F-actin. An hypothesis was proposed that F-actin may be imparting a degree of structural inflexibility to the koala sperm plasma membrane; hence, exposure of spermatozoa to cytochalasin D (5 microM), a F-actin depolymerisation agent, should result in increased plasticisation of the membrane and greater tolerance of cell volume changes that typically occur during cryopreservation. In experiment 1, koala (n = 4) and wombat (n = 4) spermatozoa packaged in 0.25 mL straws were cryopreserved using two freezing rates (fast-3 cm above liquid N2 interface; slow-6 degrees C/min in a freezing chamber) and two glycerol concentrations (8 and 14% v/v) in a tris-citrate glucose buffer with 15% (v/v) egg yolk. Wombat spermatozoa showed better (P < 0.01) post-thaw survival (% motile, % intact plasma membranes, % decondensed sperm heads) than koala spermatozoa. When exposed to media of varying osmolality, koala spermatozoa were less tolerant (% intact plasma membrane) of hyper-osmotic conditions (920 and 1410 mOsmol/kg) than wombat spermatozoa. F-actin was localised using a monoclonal antibody but only found in the wombat sperm head. When koala and wombat spermatozoa were exposed to media of varying osmolality, cytochalasin D had no beneficial effect on sperm survival (% intact plasma membranes). This study has demonstrated that wombat spermatozoa are highly tolerant of cryopreservation when compared to koala sperm but that spermatozoa from both species show greatest post-thaw survival when frozen slowly in 14% glycerol. Koala sperm are also particularly susceptible to hyper-osmotic environments but lack of detectable F-actin in the koala spermatozoan suggests that poor cryopreservation success in this species is unlikely to be associated with F-actin induced plasma membrane inflexibility.
authors.affiliation: School of Animal Studies, University Of Queensland, Gatton 4343, Australia. s.johnston1@uq.edu.au
authors.forename: S D
authors.initials: SD
authors.lastname: Johnston
authors.forename: C
authors.initials: C
authors.lastname: MacCallum
authors.forename: D
authors.initials: D
authors.lastname: Blyde
authors.forename: R
authors.initials: R
authors.lastname: McClean
authors.forename: A
authors.initials: A
authors.lastname: Lisle
authors.forename: W V
authors.initials: WV
authors.lastname: Holt
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology*; D015638:Cytochalasin D / Q000494:pharmacology; D008297:Male; D008394:Marsupialia; D009994:Osmolar Concentration; D009995:Osmosis; D048368:Phascolarctidae; D012662:Semen Preservation / Q000379:methods*; D013094:Spermatozoa / Q000473:pathology*; D013997:Time Factors
chemical_list: D000199:Actins; D003451:Cryoprotective Agents; D015638:Cytochalasin D