title: Understanding Poor Reproduction in the Maned Wolf (Chrysocyon brachyurus) Held Ex Situ
Creators: name: Reiter, Lauren E.
Publisher: name: George Mason University
Year: 2012
Subjects: subject: Maned wolf
subject: Fecal metabolites
subject: Reproduction
subject: FOS: Medical biotechnology
subject: Ovarian activity
subject: Leptin
subject: Urinary steroids

description: Maned wolves maintained in U.S. zoos have experienced limited reproductive success and high neonatal mortality. Sub-optimal health including, cystinuria, intestinal disorders and other health problems, coupled with stress associated with captive management have been indicated as potential causes of poor reproduction in the North American ex situ population. The objectives of this study were to: 1) utilize non-invasive endocrine monitoring to assess reproductive and stress hormones in female maned wolves of various reproductive histories and 2) develop and validate a urinary leptin assay as a biomarker for nutritional status in this species. For the first objective, data were obtained during 39 reproductive cycles (n = 28 females), 30 that were of breeding females and the remaining cycles were of individuals housed alone. Of the 30 breeding cycles, 18 were pregnant, 10 of which resulted in births of healthy pups surviving to adulthood. The remaining 12-paired females resulted in pseudo-pregnancy. Fecal samples were collected from each animal 1-3 times per week during the breeding season (October-March) or until pups were produced, and stored at -20°C until extraction and analyses of gonadal and adrenal hormone metabolites were carried out using validated enzyme immunoassays. Differences in mean fecal steroid metabolites were determined by analysis of variance (ANOVA) followed by multiple comparisons. Mean progestagen concentrations during the pre-ovulatory phase did not differ significantly (P &gt; 0.05) among groups; however, pregnant females (raised pups: 20.0 ± 3.0 μg/g feces, lost pups: 22.5 ± 3.0 μg/g feces) had higher (P &lt; 0.05) progestagen concentrations than pseudo-pregnant (10.3 ± 1.3 μg/g feces) and unpaired (4.2 ± 0.5 μg/g feces) counterparts during the peri-ovulatory phase. During the luteal phase, the highest (P &lt; 0.05) mean progestagen metabolite concentration was found in pregnant females that raised pups (45.8 ± 2.3 μg/g feces), followed by pregnant wolves that lost pups (43.0 ± 4.0 μg/g feces) and, then, pseudo-pregnant individuals (27.5 ± 1.2 μg/g feces). Profiles of singleton female maned wolves displayed low progestagen concentrations throughout the breeding season (mean ± SEM; 4.6 ± 0.3 μg/g feces) and the hormone level was lower (P &lt; 0.05) than those of other groups. Mean fecal cortisol concentrations during the pre-ovulatory and peri-ovulatory phases did not differ significantly (P &gt; 0.05) among groups; however, females that cared for their pups (1.8 ± 0.1 μg/g feces) had lower (P &lt; 0.05) corticoid concentrations during the luteal phase than pseudo-pregnant females (3.3 ± 0.2 μg/g feces) and individuals that experienced neonatal loss (3.1 ± 0.3 μg/g feces). Furthermore, females who successfully reared their young excreted the lowest corticoid concentrations across all three reproductive phases of the cycle. For the second objective, fresh urine (34 samples from 20 maned wolves) was opportunistically collected during the 2009-10 and 2009-11 breeding seasons. Additionally, 10 serum samples from 10 individuals were collected during routine physical examinations. Urinary and serum leptin concentrations were assessed using multi-species leptin radioimmunoassay (RIA) kit. Curves of serially diluted pooled maned wolf serum and urine exhibited parallel displacement to that of the standard curve (1.56-50 ng/mL) demonstrating immunoactivity of endogenous antigen similar to the assay standards. Net recovery of 1.56, 3.13, 6.25, 12.5, 25 and 50 ng leptin added to 200 μL neat urine was 1.12, 0.98, 1.72, 1.97, 3.86 and 8.46 ng, respectively (y = 0.31x + 0.51, R2 = 0.98) and indicated a gross underestimation of hormone mass. Leptin concentrations were correlated with biological measures (i.e., age, sex, body weight, etc.) obtained opportunistically. Relationships between serum/urinary leptin and body condition were determined using correlation analyses. Sex did not influence (t17 = -0.52, P &gt; 0.05) urinary leptin concentration in the maned wolf (males: 2.30 ± 0.44 ng/mg Cr, range: 0.35-4.29 ng/mg Cr, n = 7; females: 2.63 ± 0.41 ng/mg Cr, range: 0.80-5.32 ng/mg Cr, n = 12). Urinary leptin concentrations increased with age for both males and females (t5 =-2.98; P = 0.03). Body length showed a marginally significant positive correlation with urinary leptin concentration (r3 = 0.73, P = 0.09, n = 5); however, no other significant correlations were found with the remaining biological measures. This study demonstrated that: 1) ovulation only occurs in females paired with a male, indicating that the maned wolf is an induced ovulator; 2) pseudo-pregnant females, on average, excrete lower progestagen metabolites than pregnant individuals; 3) females that lost pups or did not become pregnant exhibit elevated corticoid excretion compared to individuals producing and raising pups; and 4) urinary leptin can be detected using a multi-species leptin RIA, although there appears to be a gross underestimation of hormone mass. These findings advance understanding about the causes of poor reproductive success in captive maned wolves, which is critical for developing an improved management strategy for ex situ populations. Future studies should focus on identifying factors contributing to and alleviating stress responses in captive maned wolves, which may then help improve pregnancy rates and reduce neonatal loss. Additional studies are also required to further explore the usefulness of urinary leptin as a biomarker for assessing nutritional status and overall health of maned wolves kept ex situ.