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An investigation into the similarities and diVerences governing the cryopreservation success of koala (Phascolarctos cinereus: goldfuss) and common wombat (Vombatus ursinus: shaw) spermatozoa

S.D. Johnston a,¤, C. MacCallum a,b, D. Blyde b, R. McClean a, A. Lisle c, W.V. Holt d

a School of Animal Studies, University Of Queensland, Gatton 4343, Australia

b Western Plains Zoo, Dubbo 2830, Australia

c School of Agronomy and Horticulture, The University of Queensland, Gatton 4343, Australia

d Institute of Zoology, The Zoological Society of London, Regent’s Park, London NW1 4RY, UK

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 inXexibility to the koala sperm plasma membrane; hence, exposure of spermatozoa to cytochalasin D (5M), 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 (nD4) and wombat (nD4) spermatozoa packaged in 0.25mL straws were cryopreserved using two freezing rates (fast—3cm above liquid N2 interface; slow—6°C/min in a freezing chamber) and two glycerol concentrations (8 and 14% v/v) in a tris–citrate glucose buVer 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 1410mOsmol/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 beneWcial eVect 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 inXexibility.

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