CSIRO PUBLISHING www.publish.csiro.au/journals/rfd Reproduction, Fertility and Development, 2009, 21, 351–363 The effect of the breeding season, cryopreservation and physiological extender on selected sperm and semen parameters of four ferret species: implications for captive breeding in the endangered black-footed ferret G. van der Horst A,C , R. M. Kitchin B , M. van der Horst A and R. W. Atherton B A Department of Medical Biosciences, University of the Western Cape, Private Bag X17, Bellville 7537, South Africa. B Department of Zoology and Physiology, University of Wyoming, Box 3166, Laramie, WY 82071, USA. C Corresponding author. Email: gvdhorst@uwc.ac.za Abstract. In the present investigation, comparative baseline information on selected sperm characteristics of ejaculate spermatozoa of the domestic (Mustela putorius furo), fitch (Mustela sp.) and black-footed ferrets (Mustela nigripes) and the Siberian polecat (Mustela eversmanni) are presented. The main emphasis was to establish differences and similarities among these species in relation to semen and sperm quality during the breeding season, in cryopreservation success and in supporting sperm motility in different extenders or physiological media. The results confirm that most sperm morphology abnormalities were evident during the beginning of the breeding cycle in all four species. No significant interspecies differences were apparent in the sperm attributes examined, for all sampling months during the breeding season. Moreover, all species exhibited comparable patterns of reproductive seasonality. Cryopreservation suppressed sperm characteristics equally in all species studied. Ejaculate spermatozoa of closely related ferret species shared many similar motion characteristics using computer-aided sperm motility analysis. These results suggest that the basic sperm physiology of the ferret species under examination is very similar. Disparate to the interspecies comparisons, there were significant differences for most sperm motion parameters when spermatozoa of any of the ferrets were compared in different extenders. Assisted reproductive technologies developed for use in domestic ferret, fitch ferret or Siberian polecat may be successfully applied to captive breeding of the black-footed ferret using semen during any of the functional breeding months. Additional keywords: domestic ferret, fitch ferret, reproductive biotechnologies, Siberian polecat, surrogate species. Introduction The black-footed ferret, Mustela nigripes, is a small, solitary, nocturnal mustelid that historically ranged from Canada toTexas (Honacki et al. 1982; Forrest et al. 1988; Clapperton 1989). However, due largely to the eradication of their primary prey base (viz. the prairie dog, Cynomys sp.), M. nigripes’ numbers declined rapidly, and by 1979 they were thought to be extinct (Anderson et al. 1986; Forrest et al. 1988). In 1981, a small population was discovered near Meeteetse, Wyoming.The Mee- teetse population grew to almost 130 animals by 1984, until an epizootic form of canine distemper almost destroyed the popula- tion in 1985 (Thorne 1986; Thorne and Williams 1988; Williams et al. 1988). The 18 remaining survivors of the epidemic were captured between 1985 and 1987 and formed the nucleus of a captive breeding program (Miller et al. 1988). The ultimate objective was to re-establish a wild population of 1500 breeding adults in at least 10 different populations in former M. nigripes habitat by the year 2010 (US Fish and Wildlife Service 1988). The captive breeding program has met with notable success and by 1998 there was a captive population of ∼300 animals (including a breeding population of 240 animals), and more than 300 animals had been reintroduced into the wild (D. Singleton- Olson, pers. comm. to R. W. Atherton). Nevertheless, biologists are not optimistic about M. nigripes’ chances of full recovery in the wild; its primary prey base is being destroyed by sylvatic plague and agricultural practices, black-footed ferrets appear to be susceptible to the sylvatic plague present in their prey, there is concern over the ability of captive-reared animals to adapt to the wild and there is uncertainty over future funding for the captive propagation of this species (Oakleaf 1989; Gober and Smith 1995). Consequently, M. nigripes remains in danger of extirpation, and can still legitimately be considered one of North America’s most endangered mammals. As part of the captive breeding program, a major research effort was initiated to sample, characterise and cryopreserve semen of all seven black-footed ferret founder males. In addition, due to the risk of losing more individuals and further depleting the already depauperate gene pool, the identification of potential © CSIRO 2009 10.1071/RD08075 1031-3613/09/020351