Changes in urinary testosterone and corticosterone metabolites during short-term confinement with repeated handling in wild male cane toads (Rhinella marina) Edward J. Narayan A,D , Frank C. Molinia B , John F. Cockrem C and Jean-Marc Hero A A Environmental Futures Centre, School of Environment, Griffith University, Gold Coast campus, Qld 4222, Australia. B Landcare Research, Private Bag 92170, Auckland 1142, New Zealand. C Institute of Veterinary, Animal and Biomedical Sciences, Massey University, Palmerston North, New Zealand. D Corresponding author. Email: e.narayan@griffith.edu.au Abstract. Stressors generally decrease testosterone secretion and inhibit reproduction in animals. Urinary testosterone and corticosterone metabolite concentrations were measured in adult male cane toads (Rhinella marina) at the time of capture from the wild and during 24 h of confinement with repeated handling. Mean urinary testosterone concentrations increased 2 h after capture, were significantly elevated above initial concentrations at 5 h, and then declined. Mean testosterone concentrations remained elevated 24 h after capture. Mean urinary corticosterone concentrations increased after capture, were significantly elevated above initial concentrations at 2 h, and remained elevated thereafter. This is the first report in amphibians of an increase in testosterone excretion after capture from the wild, with previous studies showing either no change or decline in testosterone. This finding may be associated with the mating strategy and maintenance of reproductive effort in the cane toad, a species that shows explosive breeding and agonistic male–male interactions during breeding. The finding that testosterone excretion increases rather than decreases after capture in male cane toads shows that it should not be generally assumed that reproductive hormone secretion will decrease after capture in amphibians. Additional keywords: amphibian, capture, explosive breeding, non-invasive, stress. Received 11 August 2011, accepted 17 February 2012, published online 13 March 2012 Introduction Stress can decrease or inhibit reproductive function in vertebrates (Moore and Miller 1984). Physical and psychological stimuli, known as stressors, activate the hypothalamo-pituitary- interrenal axis of amphibians and cause increased secretion of glucocorticoid hormones, such as corticosterone (Narayan et al. 2010b). It has been suggested that the activity of the gonadotropin-releasing hormone, which is responsible for the secretion of the gonadotropins, follicle-stimulating hormone, and luteinising hormone (LH), is lowered during short-term stress responses in amphibians (Moore and Zoeller 1985). However, there are some indications of increases in LH and testosterone during short-term stress responses in male birds (Heiblum et al. 2000) and particularly in dominant male birds during the stress of social conflict (Knol 1991). Decreased testosterone concentrations at the same time as increased corticosterone concentrations in response to short-term capture and handling have been reported in several amphibians (Leboulenger et al. 1979; Paolucci et al. 1990; Gobbetti and Zerani 1996; Mosconi et al. 2006; Woodley and Lacy 2010). Experimental elevation of corticosterone, such as the injection of water frogs (Rana esculenta) with corticosterone, also led to a reduction in plasma testosterone 24 h later (Paolucci et al. 1990). There is also a report of no change in testosterone concentrations at the same time as corticosterone increased after capture in frogs (Coddington and Cree 1995). Furthermore, increase in plasma testosterone and corticosterone during breeding has been reported for some explosive-breeding amphibians such as the cane toad (Rhinella marina) and the desert spadefoot toad (Scaphiopus couchii) (Orchinik et al. 1988; Harvey et al. 1997). It is well established that short-term stressors such as capture, handling and immobilisation increase plasma corticosterone in amphibians (Greenberg and Wingfield 1987). Capture and handling (for a maximum of 5 min on each sampling occasion) also causes increased urinary corticosterone metabolite concentrations in amphibians (Narayan et al. 2011a). Urine provides a better measure of short-term changes in steroidal hormones than blood plasma because the biologically potent steroids such as corticosterone and testosterone are rendered inactive during metabolism, through subtle molecular changes and through conjugation to highly charged, side chain moieties (e.g. glucuronide or sulfate molecules) before excretion. Thus, Journal compilation Ó CSIRO 2011 www.publish.csiro.au/journals/ajz CSIRO PUBLISHING Australian Journal of Zoology, 2011, 59, 264–269 http://dx.doi.org/10.1071/ZO11070