Cryopreservation of saltwater crocodile (Crocodylus porosus) spermatozoa

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 0.3 M trehalose, 0.3 M raffinose or 0.3 M sucrose and compared with glycerol (0.3–2.7 M). Although the highest levels of mean post-thaw motility were observed following cryopreservation in 0.3 M trehalose (7.6%) and 0.3 M sucrose (7.3%), plasma membrane integrity (PI) was best following cryopreservation in 2.7 M 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 0.68 M glycerol or 0.2–0.3 M 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 1.35 M glycerol) and non-permeating (0.2 or 0.3 M sucrose) cryoprotectants. Spermatozoa were cryopreserved in liquid nitrogen vapour at rates of approximately −21°C min−1 (fast freeze) or −6.0°C min−1 (slow freeze). Post-thaw survival was highest with a combination of 0.2 M sucrose and 0.68 M 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.