Non-invasive monitoring of fecal androgens in spotted hyenas (Crocuta crocuta) Stephanie M. Dloniak, a, * Jeffrey A. French, b Ned J. Place, c Mary L. Weldele, c Stephen E. Glickman, c and Kay E. Holekamp a a Department of Zoology, Michigan State University, 203 Natural Sciences, East Lansing, MI 48824-1115, USA b Departments of Psychology and Biology, University of Nebraska at Omaha, Omaha, NE 68182, USA c Department of Psychology, University of California Berkeley, Berkeley, CA 94720, USA Accepted 28 August 2003 Abstract Spotted hyenas (Crocuta crocuta) exhibit an array of behavioral and morphological characteristics that set them apart from other mammals: females are heavier and more aggressive than males, and females have external genitalia that closely resemble those of the male. Because androgenic hormones might mediate the expression of these traits, androgens are of great interest in this species. Past work on circulating androgens in wild hyenas has been limited, in part because of small sample sizes. In this study we validated a non-invasive method of monitoring variation in androgens by measuring total androgen metabolites in the feces of wild and captive spotted hyenas with an enzyme immunoassay. HPLC analysis revealed multiple immunoreactive androgen metabolites in fecal extracts from both males and females. LHRH challenge in three male and two female hyenas in captivity caused an increase in fecal androgens one to three days after LHRH injection. Furthermore, presence of bone in the diet did not affect fecal androgen con- centrations in captive female hyenas. In wild spotted hyenas, time of day of fecal deposition, time elapsed between deposition and freezing of the sample, and time elapsed between freezing and extraction did not systematically affect fecal androgen concentrations. Finally, in wild hyenas, fecal androgen patterns mirrored plasma testosterone patterns in that adult immigrant males had higher concentrations than adult natal males, and pregnant females had higher concentrations than lactating females. These methods can therefore be used in future studies addressing relationships among fecal androgens, social status, reproductive state, and behavior in spotted hyenas. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Fecal androgen metabolites; Spotted hyena; Crocuta crocuta; LHRH challenge; Non-invasive 1. Introduction During fetal development, female spotted hyenas (Crocuta crocuta) are exposed to unusually high levels of androgens, and females are heavily masculinized in various aspects of their morphology and behavior (Glickman et al., 1987, 1992; Lindeque and Skinner, 1982; Licht et al., 1992). The external genitalia of fe- males closely resemble those of males in that the clitoris is elongated to form a fully erectile pseudopenis, and the vaginal labia are fused to form a pseudoscrotum (Frank et al., 1990; Matthews, 1939; Neaves et al., 1980). Al- though adult females may weigh more than adult males, the sexes are monomorphic with respect to other adult body size measurements (Hamilton et al., 1986; Kruuk, 1972; Matthews, 1939; Van Horn et al., 2003). Females are socially dominant to adult breeding males (Kruuk, 1972; Smale et al., 1993, 1997), and females are more aggressive than adult males (Frank, 1986; Hamilton et al., 1986; Monaghan and Glickman, 1992; Szykman et al., in press). Spotted hyenas live in social groups called clans. Adult male and female clan members have separate stable linear dominance hierarchies (Frank, 1986; Holekamp and Smale, 1990; Kruuk, 1972; Tilson and Hamilton, 1984), and both male and female offspring * Corresponding author. Fax: +517-432-2789. E-mail address: dloniaks@msu.edu (S.M. Dloniak). 0016-6480/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.ygcen.2003.08.011 www.elsevier.com/locate/ygcen General and Comparative Endocrinology 135 (2004) 51–61 GENERAL AND COMPARATIVE ENDOCRINOLOGY