Research Group 4: Reproduction Biology

Subjects and Projects

3. Monitoring of hormones

 

3.1. Demonstration of 2-unsaturated C19-steroids in the urine of female Asian Elephants, Elephas maximus, and its application for reproductive monitoring and prediction of parturition

Neither Asian (Elephas maximus) nor African elephant (Loxodonta africana) poulations are self-sustaining in captivity. The major problems are a too small number of breeding bulls, an increasing number of non-cycling adult females, complications around parturition, and health problems of calves during the early months of life. These factors indicate that it is necessary to improve reproductive rates. The success and failure of elephant breeding is also dependent on the availability of techniques for the assessment of reproductive activity. To improve management activities directed on reproduction it is essential to monitor time points of reproductive functions like estrous detection and the prediction of the period of parturition. A prerequisite to predict parturition are rapid endocrine techniques to monitor (daily) luteal activity. We developed a fast analytical method which combines headspace solid-phase microextraction (SPME) and gas chromatography-mass spectrometry (GC/MS) for the analyses of the luteal phase-specific substances 5a-androst-2-en-17b-ol and -17-one. Experiments on the origin of both steroids revealed that 5a-androst-2-en-17-one and probably 5a-androst-2-en-17b-ol are generated from sulfate conjugates by a thermal process.
In the mean time this method has been routinely used to predict the period of parturiton in 11 elephant cows. Alternatively daily blood samples from the ear vein were collected from trained elephant cows where a daily urine sampling regime was not possible and measurement of 5a-pregnan-3b-ol-20one were carried out. The concentrations of both analytes started to dropp down several days prior to delivery achieving baseline levels on the days of parturition. Alltogether in 19 of 20 cases parturition was successfully predicted.

Results:
Dehnhard M, Hatt J.-M., Eulenberger K, Ochs A, Strauss G: Headspace solid-phase microextraction (SPME) and gas chromatography-mass spectrometry (GC/MS) for the determination of 5a- androst-2-en-17-one and -17ß-ol in the female Asian elephant: Application for reproductive monitoring and prediction of parturition. J. Steroid Biochem. Mol. Biol. 84, 383-391, 2003.

Cooperations with the zoos of Berlin, Leipzig, Dresden, Hannover, Hamburg, Zürich, München.

3.2. Monitoring of sympathetic nervous system (SNS) activity in elephants based on measurement of urinary catecholamines

Stress induces demands to the body that are accomplished by the activation of two systems, the hypothalamic-pituitary-adrenal axis (HPA) and the sympathetic nervous system (SNS). Stressor-induced activation of the HPA axis and the SNS results in a series of neural and endocrine adaptations known as "stress response". They are responsible for allowing the body to make the necessary physiological and metabolic changes required to cope with the demands of a stressor. Up to the present the participation of the SNS in stress response of zoo and wild animals is definitely neglected. Results from farm animals revealed that different stress situations induce extensive reactions of both stress systems. We developed a method to measure adrenaline, noradrenaline and dopamine using electrochemical detection. The catecholamines were extracted from urine and separated by high-performance liquid chromatography allowing the simultaneous measurement of all three compounds. First results revealed large individual differences and day-to-day variations. As described for cortisol metabolites (see above) there is no trend towards a diurnal secretion pattern making a standardized sampling time unnecessary. The course of dopamine and luteal activity is shown in the figure below. In both animals a dopamine surge of different magnitude was measurable which was related to the beginning of luteal activity and thus to ovulation, suggesting a role of dopamine on ovulatory mechanisms.

Results:
Dehnhard, M. Characterisation of the sympathetic nervous system of Asian (Elephas maximus) and African (Loxodonta africana) elephants based on urinary catecholamine analyses. Gen. Comp. Endocrinol. 151, 274-84, 2007.

Cooperations with the zoo in Halle , Leipzig, Dresden, Hannover, Hamburg, Zürich, München and Berlin

3.3. Reproductive biology of the sand cat (Felis margarita)

Several small cat species like the sand cat (Felis margarita) are highly stress sensitive and do not regularly reproduce in captivity. Even if they do, mortality rate of kittens and incidence of infanticide is high. Practical methods for monitoring endocrine activity are essential for assessing the reproductive potential of individual animals but do not exist in the sand cat. The non-invasive approach based on fecal steroid metabolite analyses has been shown to be convenient to obtain endocrine data concerning important incidents like estrus, pregnancy and pseudopregnancy in several felid species, without the need to collect blood samples. To investigate the reproductive characteristics of the sand cat, we monitored the reproductive activity by measuring progesterone and estrogen metabolites in feces via enzyme immunoassay (EIA). Our EIA´s based on antibodies directed against 5a-pregnane-3ß-ol-20-one-3-HS-BSA (5a-P) and 1,3,5(10)-estratrien-3,17ß-diol-17-HS-BSA to measure fecal gestagens and estrogens, respectively. Soon after mating the level of fecal gestagen metabolites increased, remained on a level with distinct day-to-day fluctuations before decreasing again to basal approximately one week before parturition (51-57 days post-mating). In contrast, during pseudopregnancy, the gestagen metabolite concentrations already dropped to basal levels approximately 30 days after breeding, which is consistent with the first half of pregnancy. Thus discrimination between pregnancy and pseudopregnancy is possible by means of the analysis of an unknown fecal gestagen metabolite which has still to be characterised. Contrariwise fecal estrogens do not seem to follow follicular activity and indicate the presence of estrus. However, there were elevated estrogen levels during the second half of the pregnancy, probably caused by placental estrogens. As a perspective, an efficient estrus marker is still to be found based on the analysis of alternative analytes.

Results:
Hanschke A, Ringleb J, Dehnhard M
Non-invasive reproductive monitoring in female sand cats (Felis margarita) by analysing fecal estrogen and gestagen metabolites
Contributions to the 7th International Conference on Behaviour, Physiology and Genetics of Wildlife, Berlin, Germany, 21-24 September 2009, in press.

Cooperations with different zoos

 3.4. Comparative metabolism of testosterone and cortisol in spotted hyenas

The metabolism of both testosterone and cortisol may lead to fecal androgens as shown in radiometabolism studies with radio-labelled testosterone (T) and radio-labelled cortisol (C) in primates. Several non-invasive assays have been used to measure testosterone and cortisol metabolites in the faeces of several carnivore species. These assays are normally based on an antibody directed against the native hormone, but the possibility of cross-reactivity of an antibody directed against testosterone with fecal androgens resulting from cortisol metabolism may lead to misleading results. In the spotted hyena we showed that two different T antibody (raised against testosterone-11-HS-BSA and -3-CMO-BSA) based assays were able to monitor a distinct increase of unknown metabolites following an adrenocorticotrophic hormone (ACTH) challenge experiment highlighting the problem of the cross-reactivity of T antibody assay with glucocorticoid metabolites (see figure).
To compare the metabolism of T and C radiometabolism studies on two male spotted hyenas were carried out. Both animals received [3H]testosterone and [3H] cortisol, respectively, while anaesthetised. Fecal samples were collected from the day before application until 5 days after application. Two different strategies were pursued:

1. Characterisation of true radiolabelled T and C derived metabolites
2. Characterisation of immunoreactive metabolites. The term immunoreactive includes all those metabolites which were detected by the antibodies irrespective whether they are derived form T or C metabolism.

To characterise fecal metabolites HPLC-separations were carried out on reversed-phase RP18 columns separating fecal metabolites according to their polarity. We showed that radio-labelled testosterone was largely metabolised to polar (probably conjugated) metabolites. By contrast the T antibody predominately traced an unknown metabolite that did not overlap with the radio-labelled T peak. This indicates that the T antibody particularly detects metabolites not derived from T metabolism.  In the sheep 21 cotisol metabolites were detected in feces after infusion of radiolabelled cortisol (Möstl et al., 2002). One group had molecular weights between 302 and 308 suggesting that they might be similar to 11-oxoetiocholanolone (mw 304). Supposed, that similar cortisol metabolites may exist in hyena feces, there a recent possibility that an antibody generated against testosterone tracks cortisol metabolites with similar structures. Current investigations deal with the characterisation of C metabolites and the comparison of T and C derived fecal metabolites.
These preliminary results of the radio-labelled metabolism experiment indicate that antibody assays designed to measure T metabolites in mammalian faeces may measure non-T metabolites thereby producing false results.

Results:
Bonnani R, East M, Goymann W, Hofer H, Dehnhard M (2007): Monitoring androgens in faeces of spotted hyenas: what are we actually measuring? Contributions to the 6th Internat. Zoo & Wildlife Res. Conference on Behaviour, Physiology & Genetics, Berlin, Germany, 7-10 October 2007, p. 46.

Cooperation with research group 1
Cooperations with the zoos of Leipzig and Münster.

for further informations see research group 1 (hyena project)

  3.5. Non-invasive monitoring to study stress and reproduction in the male cheetah

This study aims to characterise adrenal and testicular activity in male cheetah to study the impact of stress on male reproductive activity. Due to species-specific differences in metabolism and gut microflora metabolites differ between species. Therefore, for each species existing methods had to be adapted or new methods had to be created for metabolite analyses (see also fields of expertise). The most important proof whether a new non-invasive method generates meaningful results or house numbers is its biological validation, demonstrating that fecal and urinary steroid metabolites are reliable indicators of gonadal and adrenal activity whilst the molecular structures of the metabolites remained unknown.
To identify the relevant steroid metabolites, a radiometabolism study will be performed. For the development of techniques for fecal steroid analysis, experiments on the metabolism of radiolabelled steroids have provided a valuable insight into the metabolism and the excretion of hormone metabolites via faeces and urine.
For biological validation experiments the application of ACTH (to stimulate adrenal activity) and GnRH (to stimulate gonadal activity) are in progress. In addition the metabolism of radiolabelled cortisol and testosterone will be studied in the near future.

Cooperation with research group 1

see also cheetah project of research group 1

   3.6. Non-invasive monitoring of hormone in the lynx: the problem to differentiate pregnancy from pseudopregnancy

The survival of many critical endangered mammal species is often depending on successful captive breeding programs which knowledge on reproductive physiology is the species. One of the world’s most endangered felid species is the Iberian lynx. Therefore we investigated, whether non-invasive monitoring of urinary and fecal hormones metabolites can be used to characterize reproductive status as an important tool for reproductive management. We used fecal and urine samples from pregnant and pseudo-pregnant Eurasian and Iberian lynxes, respectively, for our analyses. Compared to pre-mating levels fecal progesterone metabolite profiles revealed a tendency towards higher levels in pregnant and pseudo-pregnant females with no difference between both categories. Estrogen 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 showing a post-partum increase both in pregnant and pseudo-pregnant females (see figure). The results from the Eurasian lynx revealed that the measurement of fecal progesterone metabolites led to profiles dissimilar to profiles shown in other felid species. Similar results were obtained from urinary gestagen and estrogen metabolite analysis in the Iberian lynx. In this species, however, a tendency toward higher estrogen levels was observed in pregnant females.
Alltogether fecal and urinary estrogen and gestagen metabolites were unreliable for oestrus and pregnancy diagnosis in both lynx species.
Due to the presence of the placenta in pregnant females we hypothesize, that placental steroid analysis in combination with other highly sophisticated analytical techniques, like liquid chromatography mass spectrometry or urinary relaxin analysis may lead to analytical options to confirm pregnancy and to differentiate this from pseudo-pregnancy in lynx species.

Results:

Goeritz F, Dehnhard M, Hildebrandt TB, Naidenko SV, Vargas A, Martinez F, López-Bao JV, Palomares F, Jewgenow K. (2009): Non cat-like ovarian cycle in the Eurasian and the Iberian Lynx - ultrasonographical and endocrinological analyses. REPROD DOM ANIM 44 (Suppl. 2), 87-91.
Jewgenow K, A, Göritz F, Vargas A, Dehnhard M. (2009): Seasonal profiles of ovarian activity in Iberian lynx (Lynx pardinus) based on urinary hormone metabolite analyses. REPROD DOM ANIM 44 (Suppl. 2), 92-97.
Dehnhard M, Naidenko S, Frank A, Braun B, Göritz F, Jewgenow K (2008): Non-invasive monitoring of hormones: A tool to improve reproduction in captive breeding programs of rare mammals. ANIM REPROD SCI 43, (Suppl.2), 74-82.

see also our research topic feto-maternal interactions.

3.7 Scheduled project: Reproduction biology of the black-footed cat (Felis nigripes)

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