Gamete cryopreservation of Australian ‘old endemic’ rodents – spermatozoa from the plains mouse (Pseudomys australis) and spinifex hopping mouse (Notomys alexis) Kathrine M. Ferres A,B,C , Nicole O. McPherson B,D,E , Michelle Lane B,D,E,F , Hassan W. Bakos B,D , Karen L. Kind C and William G. Breed A,D,G,H A Discipline of Anatomy and Pathology, Adelaide Medical School, Faculty of Health Sciences, The University of Adelaide, SA 5005, Australia. B Discipline of Obstetrics and Gynaecology, Adelaide Medical School, Faculty of Health Sciences, The University of Adelaide, SA 5005, Australia. C School of Animal and Veterinary Sciences, Faculty of Sciences, The University of Adelaide, SA 5005, Australia. D Robinson Research Institute, University of Adelaide, Adelaide, SA 5005, Australia. E Freemasons Centre for Men’s Health, The University of Adelaide, SA 5005, Australia. F Repromed, 180 Fullarton Road, Dulwich, SA 5065, Australia. G School of Biological Sciences, Faculty of Sciences, The University of Adelaide, SA 5005, Australia. H Corresponding author. Email: bill.breed@adelaide.edu.au Abstract. Most of the Australian ‘old endemic’ rodents have greatly reduced distributions with several species now threatened with extinction. Application of assisted reproductive technology has the potential to assist in their conservation programs in at least a few species. Here we describe an attempt to cryopreserve spermatozoa from two of these species – those of the plains mouse (Pseudomys australis) and spinifex hopping mouse (Notomys alexis), which have dramatic differences in sperm morphology. Slow and rapid freezing and three different cryoprotectant media with either raffinose, glycerol and/or skim milk were used and the results compared with those of house mouse sperm, which were used as controls. Sperm morphology, motility, membrane integrity and DNA damage were determined. Prior to cryopreservation there was a higher percentage of morphologically normal, motile, P. australis sperm than in those from N. alexis. Following cryopreservation, regardless of treatment, the percentage of motile sperm was low but it was higher when raffinose with skim milk was used as a cryoprotectant than in raffinose with glycerol albeit that minimal differences in membrane integrity or DNA damage were evident. Raffinose with skim milk should thus be used as a cryoprotectant for storing sperm of these Australian rodents in the future. Additional keywords: cryostorage, hydromyine rodents, male gamete. Received 20 November 2016, accepted 6 April 2017, published online 7 July 2017 Introduction In Australia there are two groups of native rodents the more speciose of which, the ‘old endemics’ in the Subfamily Hydromyinae, have evolved a diverse array of body forms and occur in a variety of different habitats (see Aplin 2005; Breed and Ford 2007; Van Dyck and Strahan 2008 for reviews). Around 11 species in this subfamily have become extinct since the time of European settlement with the extinction rate of these Australian rodents being similar to, or even slightly higher than, that of marsupials (Burbidge et al. 2008; Woinarski et al. 2014). In addition, many of these species are now listed as either threatened, or endangered, due to their greatly reduced ranges and distributions. The most speciose genus within the hydromyines is Pseudomys with ~20 species, closely related to which are members of the genus Notomys, which have elongated hind limbs for bipedal locomotion. At the time of European settlement there were ~10 species of Notomys but five are now extinct, with two others, N. aquilo and N. fuscus, currently listed as vulnerable (see Van Dyck and Strahan 2008; Woinarski et al. 2014). Although most species of Pseudomys and Notomys are not routinely bred in captivity, laboratory colonies of two arid-zone species, the plains mouse (Pseudomys australis) and the spinifex hopping mouse (Notomys alexis), have recently been maintained in South Australia (see Watts and Aslin 1981). Much of the reproductive biology of these native rodents is generally poorly known, but laboratory studies of P. australis and N. alexis have shown that Journal compilation Ó Australian Mammal Society 2018 www.publish.csiro.au/journals/am CSIRO PUBLISHING Australian Mammalogy, 2018, 40, 76–83 http://dx.doi.org/10.1071/AM16055