Extension, osmotic tolerance and cryopreservation of saltwater crocodile (Crocodylus porosus) spermatozoa

Semen collected from 10 saltwater crocodiles (Crocodylus porosus) was used to investigate sperm in vitro manipulation and preservation. Preliminary studies revealed that phosphate buffered saline (PBS) without Ca2 +, Mg2 + and egg yolk (EY) was a suitable extender for studies of sperm physiology. Spermatozoa diluted in PBS showed no change in survival [% motility (M), rate of sperm movement (R) and % plasma membrane integrity (PI)] when diluted over a range of 1:1 to 1:16. Except for a small decline in PI, there was no change in sperm survival when semen diluted without EY was cooled rapidly to and rewarmed from 0 °C. The addition of EY (0, 5, 10 and 20% v/v) had no beneficial effect on sperm survival when incubated in PBS for 1 h at 30 °C or after 24 h storage at 4 °C. Whilst crocodile spermatozoa exposed to a range of anisotonic media and then returned to solutions of 390 mosM kg− 1 retained their M from 220 to 390 mosM kg− 1, PI remained high in hypotonic media (25-280 mosM kg −1); spermatozoa showed an increase (P < 0.05) in the incidence of flagellar coiling (FC) with increasing hypotonic conditions. The adverse effect of anisotonic conditions on sperm M and FC recovered somewhat when sperm were returned to the 390 mosM kg −1 media, but not to pre-treatment levels. Exposure of crocodile spermatozoa to respective concentrations of 0.68 M, 1.35 M and 2.7 M glycerol, dimethylsulphoxide (DMSO), and dimethylacetamide (DMA) after 2 h storage at 4 °C (equilibration) resulted in a reduction in M, but no change in PI. Sperm cryopreserved in the same cryoprotectant media within 0.25 mL straws at − 6 °C/min in a programmable freezer and thawed at 37 °C for 1 min showed a major decline (P < 0.05) of M but there was moderate protection of PI (DMA 2.7 M - 17.7 ± 4.4; DMSO 2.7 M - 22.7 ± 1.4 and glycerol 2.7 M - 25.7 ± 6.4). Sperm thawed and immediately washed to remove the cryoprotectant showed an improvement (P < 0.05) in PI but not M. Future studies of crocodile sperm preservation should explore the apparent disjunction between low levels of M and the high tolerance of the plasma membrane to anisotonic conditions and cryoprotectant toxicity.