Patterns of Testicular Activity in Captive and Wild Canada Lynx (Lynx Canadensis) ⇑ Kerry V
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General and Comparative Endocrinology 169 (2010) 210–216 Contents lists available at ScienceDirect General and Comparative Endocrinology journal homepage: www.elsevier.com/locate/ygcen Patterns of testicular activity in captive and wild Canada lynx (Lynx canadensis) ⇑ Kerry V. Fanson a,b,f, , Nadja C. Wielebnowski b, Tanya M. Shenk c, Walter J. Jakubas d, John R. Squires e, Jeffrey R. Lucas f a Department of Brain, Behaviour, and Evolution, Macquarie University, 209 Culloden Road, Marsfield, NSW 2122, Australia b Department of Conservation Science, Chicago Zoological Society, 3300 Golf Road, Brookfield, IL 60513, USA c Mammals Research, Colorado Division of Wildlife, 317 West Prospect, Fort Collins, CO 80526, USA d Maine Department of Inland Fisheries and Wildlife, 650 State Street, Bangor, ME 04401, USA e Rocky Mountain Research Station, Forestry Sciences Laboratory, 800 E. Beckwith Avenue, Missoula, MT 59808, USA f Department of Biological Sciences, Purdue University, 915 W. State Street, West Lafayette, IN 47907, USA article info abstract Article history: Canada lynx are listed as a threatened species in the contiguous US. Understanding the reproductive Received 4 May 2010 characteristics (i.e., mating system, behavior, physiology) of a species is useful for ensuring effective Revised 29 August 2010 in situ and ex situ management plans. The goal of this study was to describe patterns of androgen expres- Accepted 1 September 2010 sion in both captive and wild male Canada lynx using fecal hormone metabolite analysis. Among captive Available online 7 September 2010 lynx, juvenile and castrated males had lower concentrations of fecal androgens (fA) than intact males, thereby demonstrating that the assay detects biologically meaningful differences in testicular activity. Keywords: We found that captive males in general had much higher fA levels than wild males. All males showed Androgens strong seasonal variation in fA concentrations, with significantly higher levels being expressed during Fecal metabolites Lynx the breeding season (February and March) than during the non-breeding season. Among captive males, Non-invasive variation in seasonal fA levels did not correlate with latitude. Finally, males housed with intact cage- Seasonality mates (either male or female) had significantly higher fA levels than males housed alone or with a neu- Testosterone tered cage-mate. Ó 2010 Elsevier Inc. All rights reserved. 1. Introduction throughout the year, and also throughout their 10-year population cycle in northern populations (Poole, 2003). It is unclear whether Canada lynx (Lynx canadensis) are the primary felid species these breeding restrictions are mediated solely by changes in fe- inhabiting North America’s boreal forest. They are meso-carnivores male physiology, or whether males also exhibit physiological (9–11 kg) and feed primarily on snowshoe hares and squirrels changes that may impact reproduction. Developing a better under- (Nowak, 1999). In the northern part of their range, their depen- standing of male reproductive physiology in Canada lynx may pro- dence on snowshoe hares is so strong that their population size vide insights into how animals adapt to arctic environments, and fluctuates with snowshoe hare density in 10-year cycles (Poole, may aid in situ and ex situ conservation efforts. 2003; Ruggiero et al., 2000). Lynx are primarily found in coniferous This study integrates information from both captive and wild forests, particularly those with a mixture of forest age classes lynx populations to establish basic knowledge about the reproduc- (Poole, 2003). Lynx are solitary, but there is some overlap of home tive physiology of male Canada lynx. We used the non-invasive ranges between males and females (Ruggiero et al., 2000). South- technique of fecal hormone analysis to establish normative pat- ern populations of lynx have declined dramatically in the last terns of androgen expression in male lynx. Specifically, our objec- century, and in 2000, they were listed as a threatened species by tives were to (1) validate an assay for monitoring testicular activity the US Fish and Wildlife Service (USFWS, 2000). non-invasively, (2) characterize normative patterns of testicular One poorly understood aspect of lynx biology is their repro- activity, particularly around the breeding season, (3) compare ductive physiology. Lynx reproduction fluctuates dramatically patterns of hormone expression between captive and wild lynx, and (4) examine the effect of breeding status and housing situation on levels of fecal androgen metabolites (fA) in captive animals. This study provides the first longitudinal data on androgen levels in ⇑ Corresponding author at: Department of Brain, Behaviour, and Evolution, wild and captive Canada lynx. As such, it serves as an important Macquarie University, 209 Culloden Road, Marsfield, NSW 2122, Australia. Fax: +61 2 9850 4299. foundation for future studies and the development of more effec- E-mail address: [email protected] (K.V. Fanson). tive management plans. 0016-6480/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ygcen.2010.09.001 K.V. Fanson et al. / General and Comparative Endocrinology 169 (2010) 210–216 211 2. Methods well. Plates were read when the optical density of the maximum binding wells was >0.6 using a single filter at 405 nm using an opti- 2.1. Animals and fecal sample collection cal density plate reader (Dynex MRX Revelation, Dynex Technolo- gies, Chantilly, VA). All samples were assayed in duplicate, and data 2.1.1. Captive are expressed as ng/g wet fecal weight. This study included 28 captive Canada lynx males from 19 insti- The assay was biochemically validated for Canada lynx by dem- tutions (3 juveniles, 7 castrated males, and 18 intact, sexually onstrating (1) parallelism between serially diluted extracts and the mature males). All lynx were housed outdoors at least 50% of the standard curve, and (2) significant (>80%) recovery of exogenous time, and thus were exposed to natural photoperiod rhythms. Ani- testosterone added to fecal extracts. To monitor precision and mal care staff collected fecal samples 2–4 times per week during reproducibility, low (65% binding) and high (30% binding) quality routine cage cleanings. Most samples were collected during the control samples were run on each plate. Intra-assay coefficients breeding season (February–April), but sampling extended into of variation were 9.5% and 7.9% (n = 14) for low and high controls, other parts of the year for several individuals. When multiple lynx respectively. The inter-assay coefficients of variation were 20.2% were housed together, icing dye (Wilton Industries, Wilton, IL) was and 22.4% (n = 97), respectively. Although this degree of inter-as- delivered to each individual in a food treat, and then color was say variation is relatively high due to the extended duration of used to distinguish between feces. the study, the amount of inter-assay variation for a given individ- ual was minimized by analyzing all of their samples at the same time, whenever feasible. 2.1.2. Wild Fecal samples were collected from wild adult males in Colorado (44 individual lynx, 339 samples), Maine (7 lynx, 51 samples), and 2.3. Statistical analysis Montana (9 lynx, 39 samples). The populations in Maine and Mon- tana are naturally-occurring, while the population in Colorado has All data were analyzed using SAS 9.1 (Cary, NC) and hormone been reintroduced. All samples were collected by snow-tracking data were log transformed to meet assumptions of normality and radio-collared lynx between December and April, a time period homoscedasticity. Means provided in the text and figures are which includes their breeding season. Samples were collected from least-squares means that have been back-transformed, unless 1999 to 2008. Samples were generally collected within 24 h after otherwise noted. One captive male (M13) showed unusually low defecation, and evidence from field experiments suggests that fA fA values (similar to values observed in juveniles), even though metabolites remain stable in winter field conditions for at least he was an intact adult housed with an intact female. Therefore, four days (Fanson, 2009). Therefore, we are confident that we were he was excluded from all analyses except seasonality, in which able to obtain meaningful results from samples collected in the he was included because his general pattern of fA expression was field. To ensure that fA expression in reintroduced lynx was not af- similar to other intact males. fected by the translocation, we only included samples that had been collected at least six months post-release. 2.3.1. Status To assess the biological relevance of the assay, overall mean fA 2.2. Steroid extraction and analysis values were compared between juvenile (n = 3), castrated (n = 7), and intact, sexually mature (n = 17) males using a one-way ANO- All samples were stored in zip-lock bags at À20 °C and shipped VA. Only captive males were included, since nearly all wild males on ice to the Brookfield Zoo for processing and hormone metabolite were adults. Because two of the juveniles were housed together analysis. We extracted steroid metabolites by adding 5 ml of 80% and their samples were indistinguishable, they were combined ethanol to 0.5 g of well mixed, wet fecal material in polypropylene for statistical analysis. A Tukey–Kramer adjustment was used to tubes. Capped tubes were placed on a rotator overnight and then correct for multiple pairwise comparisons. centrifuged for 15 min at 1500 rpm. One ml of supernatant was transferred to a new polypropylene tube and diluted with 1 ml as- say buffer (0.1 M phosphate-buffered saline (PBS) containing 1% 2.3.2. Seasonality BSA, pH 7.0). Extracts were stored at -20 °C. We conducted a repeated measures ANOVA to test the effect of Fecal androgen metabolites (fA) were quantified using a single- month on fA values for both captive and wild populations. The antibody enzyme-immunoassay (EIA). Testosterone polyclonal model also controlled for the effect of year (sample collection from antibody R156/7 and the corresponding horseradish peroxidase wild lynx spanned 10 years; captive collection only spanned (HRP) conjugate were obtained from C.