Freshwater fish sperm are inactive in the male reproductive tract and seminal plasma. They are activated
under hypotonic conditions, with a brief period of motility at the time of fertilization. The aims were to find a
dilution medium that would maintain sperm inactivity, to assess the cytotoxicity of a range of
cryoprotectants, and test the effectiveness of selected diluents for cryopreservation of Murray cod sperm.
Sperm remained immotile in ionic and non-ionic solutions above 300 mOsm kg−1. Sperm motility after
centrifugation and re-suspension in fresh water was better for solutions of 300 and 600 mOsm kg−1 than
900 mOsm kg−1. In the presence of cryoprotectants, dimethylsulfoxide, dimethylacetamide, and methanol,
recovery was better at 300 mOsm kg−1 compared to 600 mOsm kg−1. Sperm diluted in all media containing
glycerol remained inactive after centrifugation and re-suspension in water. A cryopreservation diluent
composed of 300 mOsm kg−1 D-Sorbitol (DS) solution with 10% methanol produced the best post-thaw
motility (51.4±3.4%), followed by Tris–Sucrose–Potassium (TSK) solution with 10% methanol (32.9±4.7%),
and Modified Kurokuras medium with 10% methanol (27.5±4.8%). Fluorescent staining for membrane
integrity (SYBR-14 and Propidium Iodide) reflected the post-thaw motility results. Fertilization trials using
sperm frozen in DS solution with 10% methanol produced a hatch rate of 11.0% (63.1±18.23% of a control fresh
sperm hatch rate) and 6.4% (58.5±32.50% of fresh sperm hatch rate) using sperm frozen with TSK with 10%
methanol. This represents the first successful cryopreservation of sperm from Murray cod. Sperm
cryopreservation will facilitate both conservation and aquaculture of the Murray cod and related endangered
MACCULLOCHELLA species.