Cryopreservation of saltwater crocodile (Crocodylus porosus) spermatozoa S. D. Johnston A,G , E. Qualischefski A , J. Cooper B , R. McLeod C , J. Lever C , B. Nixon D , A. L. Anderson D , R. Hobbs E , J. Gosa ´lvez F , C. Lo ´ pez-Ferna ´ndez F and T. Keeley A A School of Agriculture and Food Science, The University of Queensland, Gatton, Qld 4343, Australia. B Just Genes Artificial Breeding Services, Everton Park, Brisbane, Qld 4053, Australia. C Koorana Crocodile Farm, Coowonga, Qld 4702, Australia. D School of Environmental and Life Sciences, The University of Newcastle, Callaghan, NSW 2308, Australia. E Taronga Conservation Society, Mosman, NSW 2088, Australia. F Department of Biology, Universidad Auto ´ noma de Madrid, Madrid, 20849, Spain. G Corresponding author. Email: s.johnston1@uq.edu.au 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 0.3 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 À218C min À1 (fast freeze) or À6.08C min À1 (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 Æ 4.7%; PI 20.7 Æ 2.0%) or slow (motility 12.0 Æ 2.7%; PI 22 Æ 4%) cryopreservation rate. Additional keywords: glycerol, non-permeating cryoprotectants, permeating cryoprotectants, raffinose, sucrose, trehalose. Received 18 December 2016, accepted 21 February 2017, published online 30 March 2017 Introduction Similar to what has been achieved for domestic animal pro- duction species, the use of frozen–thawed spermatozoa com- bined with AI has the potential to delivery major benefits to the global farmed crocodile industry. These benefits include accelerating the rate of genetic development and production gain, reducing the requirement for keeping males on-farm and allowing for the preservation of valuable genetics through time and space (Johnston et al. 2014a). Successful cryopreservation technology will also be essential in establishing genome banks (Holt et al. 2003) for the genetic management of rare and endangered crocodilian species (Clulow and Clulow 2016). Although studies of reptilian sperm cryopreservation have been extremely limited (Browne et al. 2011; Clulow and Clulow 2016; Young et al. 2017), the ability to collect comparatively large-volume ejaculates (,1 mL) of highly concentrated, uncontaminated motile spermatozoa from the saltwater croco- dile (Johnston et al. 2014b) using non-lethal methods has facilitated the opportunity for a research model that allows a more systematic and empirical assessment of reptilian gamete physiology and thus inform improvements in cryopreservation strategies. Molinia et al. (2010) have also established a repro- ductive model species for developing assisted breeding technol- ogy for rare and endangered New Zealand lizards, but to date this has not extended to the cryopreservation of spermatozoa. In previous studies, Johnston et al. (2014c) showed that the saltwater crocodile sperm membrane is highly tolerant of both extreme hypotonic media and different concentrations (0.68, CSIRO PUBLISHING Reproduction, Fertility and Development http://dx.doi.org/10.1071/RD16511 Journal compilation Ó CSIRO 2017 www.publish.csiro.au/journals/rfd