Martin Dehnhard

Does anal gland scent signal identity in the spotted hyaena

Recognition requires the existence of distinct cues and the ability to detect and discriminate between these signals. Using gas chromatography–mass spectrometry, we investigated the chemical composition of 143 anal scent gland secretions from 84 individually known spotted hyaenas, Crocuta crocuta, in three clans in the Serengeti National Park, Tanzania. In total, 252 volatile compounds were detected. Comparison of the chemical composition of individual scent profiles by calculating the Bray–Curtis similarity index and using an analysis of similarities revealed that scent profiles varied significantly between individuals but also changed in composition over time. There was no difference in complexity, in terms of the number of different compounds, between male and female scent or between scent from high- and low-ranking females. Scent profiles of closely related females were as similar as profiles of unrelated females. Although there was no evidence of rank-related changes in profiles across the entire female social hierarchy, the profiles of high-ranking females differed significantly from those of low-ranking females, suggesting the presence of an olfactory badge of status. Behaviourally, adult females responded to scent deposited on vegetation by other females significantly faster and more often, and were more likely to anoint themselves with that scent, as the social rank of the female who had deposited the scent increased. These results suggest that spotted hyaena scent carries both an olfactory badge of status and individually distinct cues that are likely to be useful in the maintenance of a social network within their fission–fusion society.

The use of noninvasive and minimally invasive methods in endocrinology for threatened mammalian species conservation.

Endocrinology is an indispensable tool in threatened species research. The study of endocrinology in threatened species not only advances knowledge of endocrine mechanism but also contributes to conservation efforts of studied species. To this end, endocrinology has been traditionally used to understand reproductive and adrenocortical endocrine axes by quantifying excreted steroid metabolites. From these studies a large body of knowledge was created that contributed to the field of endocrinology, aided conservation efforts, and created a template by which to validate and conduct this research for other species. In this regard noninvasive hormone monitoring has become a favored approach to study the basic endocrinology of wildlife species. Due to the increased understanding of endocrine physiology of threatened species, breeding rates of captive population have improved to levels allowing for reintroduction of species to restored natural ecosystems. Although these approaches are still employed, advances in biochemical, molecular, and genomic technologies are providing inroads to describe lesser known endocrine activity in threatened species. These new avenues of research will allow for growth of the field with greater depth and breadth. However, for all approaches to endocrinology, limitations on resources and access to animals will require innovation of current methodologies to permit broad application for use in threatened species research.

Non-invasive Monitoring of Hormones: A Tool to Improve Reproduction in Captive Breeding of the Eurasian Lynx

The survival of many critical endangered mammal species is often depending on successful captive breeding programmes which include the future option of reintroduction to the wild. Breeding in captivity also demands the application of modern assisted reproductive techniques to ensure maximal biodiversity, but knowledge on reproductive physiology is often limited. Therefore, non-invasive monitoring of urinary and faecal hormones has become an important tool for reproductive management. To exemplify the importance of non-invasive hormone monitoring, we choose the Eurasian lynx as a model for the worlds most endangered felid species, the Iberian lynx. We analysed faecal samples of pregnant and pseudo-pregnant female Eurasian lynxes during a 3-year study period. Compared to pre-mating levels faecal progesterone metabolite profiles revealed a tendency towards higher levels in pregnant and pseudo-pregnant females with no difference between both categories. Oestrogen levels raised in both pregnant and pseudo-pregnant females with a tendency to be more elevated and prolonged in pregnant females. Surprisingly both E2 and P4 metabolites were highly correlated (r(2) =0.8131, p < 0.0001) showing a postpartum increase both in pregnant and pseudo-pregnant females. The results from the Eurasian lynx revealed that the measurement of faecal progesterone metabolites led to profiles dissimilar to profiles shown in other felid species, but similar to those from faecal gestagen metabolite analysis in the Iberian lynx. To identify faecal gestagen and oestrogen metabolites a radio-metabolism study was performed. Using the progesterone immunoassay two major progesterone metabolites were detected demonstrating that the assay indeed tracks the relevant metabolites. The oestrogen assay measured authentic 17beta-oestradiol and oestrone, and their conjugates. The analysis of the faecal metabolite composition in samples from early and late pregnancy and lactation particularly revealed a distinct shift in the relation between 17beta-oestradiol and oestrone that changed in favour of oestrone. This might indicate different hormone sources during and after pregnancy (corpus luteum, placenta). We hypothesize, that placental steroid analysis in combination with other highly sophisticated analytical techniques, like liquid chromatography mass spectrometry or urinary relaxin analysis may led to analytical options to confirm pregnancy and to differentiate this from pseudo-pregnancy in lynx species.

Non cat-like ovarian cycle in the Eurasian and the Iberian lynx - ultrasonographical and endocrinological analysis.

The Iberian lynx is considered the most endangered felid species. Therefore, an ex situ conservation program was initiated to protect this species from extinction. Additional knowledge on lynx reproduction biology and reliable methods for reproductive monitoring are important for developing a captive breeding program. The aim of this study in lynx was to implement transrectal ultrasonography to visualize ovarian structures (follicles, corpora lutea) and to assess ovarian activity in addition to analysis of serum progesterone and oestradiol. Because of limited access to Iberian lynxes, the less-endangered Eurasian lynx and bobcat were also studied in this comparative study. Recent endocrinological studies based on faecal and urinary progesterone and oestrogen metabolites revealed that steroid profiles in both these species were alike and did not follow the typical pattern of other felids. Pregnancy diagnosis was not possible, since progesterone concentrations did not differ between pregnant and pseudopregnant animals. Progesterone was also detected after parturition as well as after weaning until the onset of a new oestrous cycle. In the present study, the presence of corpora lutea during the non-breeding season was confirmed by ultrasonography and by elevated serum levels of progesterone averaging 3.56 +/- 1.3 ng/ml in Eurasian and 6.1 +/- 0.26 ng/ml in Iberian lynx, respectively. The ultrasonographical findings on the ovarian structures suggest strongly that corpora lutea developed after ovulation stay active until November and regress before the onset of the next oestrus.

Pregnancy diagnosis in urine of Iberian lynx (Lynx pardinus)

Diagnosis of pregnancies is an important management tool for the Iberian lynx Conservation Breeding Program, a program geared to recover the worlds most endangered felid. Non-invasive methods such as fecal hormone analyses are not applicable to the lynx, since fecal progestin does not follow the typical pregnancy pattern of felids. Therefore, we aimed to test whether urine can be used as an alternative substance for pregnancy diagnosis in the Iberian lynx. Progesterone immunoreactive metabolites were determined in urine samples of pregnant and non-pregnant females before and during breeding season. Additionally, we used the Witness Relaxin test to determine relaxin in blood and urine. No differences were found in progestin concentrations determined in urine samples collected from pregnant and non-pregnant animals between day 1 and 65 following mating. Although the Witness Relaxin test was positive in serum samples collected from animals between day 32 and 56 of pregnancy, it failed in both fresh and frozen urine samples collected from the same stage of pregnancy. A weak relaxin reaction in urine samples collected from animals between day 29 and 46 of pregnancy was detectable after urines were concentrated by ultrafiltration (>50x). Concentrated samples obtained from non-pregnant and early pregnant animals yielded negative test results. In conclusion, the Witness Relaxin test can be applied for pregnancy diagnosis in Iberian lynx in both serum and concentrated urine samples obtained during the second half of pregnancy. A positive relaxin test indicates an ongoing pregnancy, whereas negative tests must be judged carefully as hormone concentrations might be below detection thresholds.

PGFM (13,14-dihydro-15-keto-PGF2α) in pregnant and pseudo-pregnant Iberian lynx: A new noninvasive pregnancy marker for felid species

In mammals, uterine and placental prostaglandin F(2alpha) is involved in the regulation of reproduction-related processes such as embryonic development, initiation of parturition, and resumption of ovarian activity. Prostaglandin F(2alpha) (PGF(2alpha)) is rapidly metabolized to its plasma metabolite PGFM (13,14-dihydro-15-keto-PGF(2alpha)), which has also been detected in urine. Therefore, the current study aimed to develop and validate an efficient, quick, and inexpensive enzyme immunoassay (EIA) for PGFM estimation in urine of the Iberian lynx (Lynx pardinus) for pregnancy monitoring and for differentiation between pregnancy and pseudo-pregnancy. Urine samples collected from captive Iberian lynx (11 pregnant and 4 pseudo-pregnant cycles) were subjected directly to a PGFM EIA. The assay was validated for parallelism, precision, and stability of urinary PGFM. In addition, high-performance liquid chromatography (HPLC) immunograms and liquid chromatography-mass spectrometry (LCMS) were performed to identify PGFM within urine samples. Urinary PGFM levels before mating and after parturition were about 1.5 ng/mL. After Day 20 postmating, both pregnant and pseudo-pregnant females showed slight increase of hormone levels; in pseudo-pregnant females, this elevation did not exceed 7 ng/mL. A significant increase in pregnant females was observed after Day 45 postmating; urinary PGFM increased from 10 ng/mL at Day 45 toward a peak of 46.0+/-19.3 ng/mL around parturition. First results show that PGFM is detectable in feces as well and follows similar courses as shown for urine. In conclusion, the presented and validated PGFM assay is an easy and reliable method for noninvasive pregnancy diagnosis in the Iberian lynx (and probably other felids) if applied approximately 20 d prior parturition in pure urine or fecal extracts. High PGFM levels in urine or fecal samples may allow a pregnancy diagnosis without knowledge of mating time, making the PGFM test applicable to free-ranging animals.

Using PGFM (13,14-dihydro-15-keto-prostaglandin F2α) as a non-invasive pregnancy marker for felids

Understanding the complex endocrine interactions that control reproduction in felids is essential for captive breeding management. The most important demand is a quick and reliable pregnancy diagnosis. However, the occurrence of pseudopregnancies in felids complicates matters. We investigated whether the fecal prostaglandin metabolite (PGFM) recently suggested for pregnancy diagnosis in the lynx is suitable for all felid species. We found that increased levels of PGFM during the last trimester indicate pregnancy in seven of the eight main lineages of the carnivore family Felidae. PGFM levels in a sand cat (domestic cat lineage) were basal at mating and remained so until Day 40 post-mating. Day 41 marked the beginning of a distinct increase culminating in peak levels of 6.5 μg/g before parturition and decreasing again to baseline thereafter. Similar pregnancy profiles were obtained from the domestic cat, the leopard cat, the lynx, the ocelot and the caracal lineage, whereas in pseudopregnant individuals (sand cat, Iberian and Eurasian lynx) fecal PGFM remained at basal levels. In pregnant cheetahs (puma lineage) PGFM increased above basal following day ∼48 peaking before pregnancy but remained at baseline in pseudopregnant females. Discrepancies existed in the Panthera lineage. While Chinese leopard, Sumatran tiger, and the black panther showed marked increases of PGFM during the last weeks of pregnancy, only moderate increases in PGFM levels were found in the Indochinese tiger and the Persian leopard. Altogether, PGFM as tool for pregnancy diagnosis has been proven to be useful in breeding management of felids.

Plasma testosterone levels decrease after activation of skin immune system in a free-ranging mammal

The immunocompetence handicap hypothesis (ICHH) postulates a trade-off between the display of androgen-related, sexually selected traits and investment in immune functions, often summarized as immunosuppression by testosterone (TE). We investigated the relationship between delayed type hypersensitivity (DTH) skin responses and the endocrine system in a free-ranging mammal and asked: (i) are high plasma TE levels related to a weak DTH response or (ii) does an activation of the cellular immune system cause a decrease in plasma TE secretion? and (iii) does baseline cortisol (CORT) and the capacity to release CORT in response to stress influence the DTH response? We studied these questions in the bat Carollia perspicillata and conducted an immune challenge (IC), a DTH skin test with the antigen 1,4-dinitrofluorobenzene (DNFB), as a measure for antigen-specific cell-mediated immunity. We found a high individual variance in DTH response irrespective of sex. In addition, pre-IC plasma TE did not affect the extent of DTH responses, but instead the IC lowered post-IC TE titres in both sexes. Also, pre-IC baseline CORT titres did not influence the DTH responses significantly, but the extent of an induced CORT response was negatively related to the DTH reaction. Our study does not support the presumption of an immunosuppressive effect of TE, but suggests that an activation of the immune system reduces plasma TE. This is still in line with the ICHH as originally formulated, because only immunocompetent animals are able to cope with pathogens effectively and maintain high TE levels at the same time.

Comparative metabolism of gestagens and estrogens in the four lynx species, the Eurasian (Lynx lynx), the Iberian (L. pardinus), the Canada lynx (L. canadensis) and the bobcat (L. rufus).

With the increasing prevalence of faecal hormone metabolite analysis, it is important to develop a better understanding of the dynamics of faecal metabolite composition. The aim of this study was to compare the quantitative faecal gestagen and estrogen metabolite composition in the four lynx species: Eurasian lynx, Iberian lynx, Canada lynx and bobcats. Comparative HPLC immunograms were generated from faecal samples collected before, during, and after pregnancy from individual females of each lynx species. Gestagens and estrogens revealed three similar classes of immunoreactive faecal metabolites: (1) polar metabolites which were enzyme-hydrolysable and thus may be designated as conjugates, (2) non-hydrolysable polar metabolites, and (3) non-polar metabolites or free steroids. For both hormones, strong similarities in the HPLC immunograms across species suggests that steroid metabolism is relatively conserved among Lynx species. Gestagens were primarily excreted as polar conjugates or unknown metabolites, whereas estrogen metabolism revealed a huge proportion (approximately 50%) consisting of 17beta-estradiol and estrone. These results are consistent with patterns of steroid metabolism in other felid species. Only two minor species-specific patterns emerged. In bobcats, we observed an exceptionally high proportion of gestagen conjugates, and in Iberian lynx, there was an exceptionally high proportion of estrone. The comparison of HPLC immunograms within individuals revealed that intra-individual variations in steroid metabolite composition are considerably high. However, changes in metabolite composition did not correlate with specific reproductive stages; rather, they seemed to occur at random. We assume that these differences may reflect changes in liver metabolism and/or qualitative and quantitative variations in gut bacteria composition, resulting in differences in faecal metabolite composition.

Progesterone and estradiol in cat placenta--biosynthesis and tissue concentration.

Ovarian and placental steroids are essential for the maintenance of pregnancy. In some mammals it is evident that the placenta is responsible for the production of steroids. However, in the domestic cat, steroid secretion from the placenta has not yet been elucidated. Our study aimed to find out whether feline placentae are able to produce steroids. Placentae from different pregnancy stages were analyzed for mRNA expression of five steroidogenic enzymes (HSD3B1, CYP11A1, CYP17A1, HSD17B1 and CYP19A1) and for tissue concentrations of progesterone and estradiol. Steroidogenic enzymes responsible for the final steps of estradiol (CYP19A1) and progesterone synthesis (HSD3B) were expressed at very high levels and followed almost the same pattern over pregnancy as the intraplacental hormones themselves. By contrast, the other enzymes were found in very low quantities suggesting that biosynthesis occurs via extra-placental steroid precursors. The plasma steroid profiles measured by other groups differ from the placental hormone courses determined by us; therefore we conclude that the feline placenta can produce progesterone and estradiol.