title: Cryopreservation of saltwater crocodile (Crocodylus porosus) spermatozoa.
abstract: The aim of the present study was to develop a protocol for the successful cryopreservation of Saltwater crocodile spermatozoa. Sperm cells were frozen above liquid nitrogen vapour in phosphate-buffered saline (PBS) containing either 0.3M trehalose, 0.3M raffinose or 0.3M sucrose and compared with glycerol (0.3-2.7M). Although the highest levels of mean post-thaw motility were observed following cryopreservation in 0.3M trehalose (7.6%) and 0.3M sucrose (7.3%), plasma membrane integrity (PI) was best following cryopreservation in 2.7M glycerol (52.5%). A pilot study then assessed the cytotoxicity of glycerol and sucrose prior to cryopreservation and revealed no loss of survival when spermatozoa were diluted in 0.68M glycerol or 0.2-0.3M sucrose once cryoprotectants were washed out with PBS or Biggers, Whitten and Whittingham medium containing sperm capacitation agents (BWWCAP). A final study refined the combined use of permeating (0.68 or 1.35M glycerol) and non-permeating (0.2 or 0.3M sucrose) cryoprotectants. Spermatozoa were cryopreserved in liquid nitrogen vapour at rates of approximately -21°Cmin-1(fast freeze) or -6.0°Cmin-1(slow freeze). Post-thaw survival was highest with a combination of 0.2M sucrose and 0.68M glycerol and when these cryoprotectants were washed out with BWWCAP, regardless of whether spermatozoa were frozen using a fast (motility 14.2±4.7%; PI 20.7±2.0%) or slow (motility 12.0±2.7%; PI 22±4%) cryopreservation rate.
authors.affiliation: School of Agriculture and Food Science, The University of Queensland, Gatton, Qld 4343, Australia.
authors.forename: S D
authors.initials: SD
authors.lastname: Johnston
authors.affiliation: School of Agriculture and Food Science, The University of Queensland, Gatton, Qld 4343, Australia.
authors.forename: E
authors.initials: E
authors.lastname: Qualischefski
authors.affiliation: Just Genes Artificial Breeding Services, Everton Park, Brisbane, Qld 4053, Australia.
authors.forename: J
authors.initials: J
authors.lastname: Cooper
authors.affiliation: Koorana Crocodile Farm, Coowonga, Qld 4702, Australia.
authors.forename: R
authors.initials: R
authors.lastname: McLeod
authors.affiliation: Koorana Crocodile Farm, Coowonga, Qld 4702, Australia.
authors.forename: J
authors.initials: J
authors.lastname: Lever
authors.affiliation: School of Environmental and Life Sciences, The University of Newcastle, Callaghan, NSW 2308, Australia.
authors.forename: B
authors.initials: B
authors.lastname: Nixon
authors.affiliation: School of Environmental and Life Sciences, The University of Newcastle, Callaghan, NSW 2308, Australia.
authors.forename: A L
authors.initials: AL
authors.lastname: Anderson
authors.affiliation: Taronga Conservation Society, Mosman, NSW 2088, Australia.
authors.forename: R
authors.initials: R
authors.lastname: Hobbs
authors.affiliation: Department of Biology, Universidad Autónoma de Madrid, Madrid, 20849, Spain.
authors.forename: J
authors.initials: J
authors.lastname: Gosálvez
authors.affiliation: Department of Biology, Universidad Autónoma de Madrid, Madrid, 20849, Spain.
authors.forename: C
authors.initials: C
authors.lastname: López-Fernández
authors.affiliation: School of Agriculture and Food Science, The University of Queensland, Gatton, Qld 4343, Australia.
authors.forename: T
authors.initials: T
authors.lastname: Keeley
mesh_terms: D000489:Alligators and Crocodiles; D000818:Animals; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000008:administration & dosage; D008297:Male; D012662:Semen Preservation / Q000379:methods*; D013081:Sperm Motility; D013094:Spermatozoa; D013395:Sucrose / Q000008:administration & dosage; D014199:Trehalose / Q000008:administration & dosage
chemical_list: D003451:Cryoprotective Agents; D013395:Sucrose; D014199:Trehalose