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SummaryStress enhances the production of corticosteroids by the adrenal cortex, resulting in the increased excretion of their metabolites in urine and faeces. An intraperitoneal injection of radioact ive corticosterone was applied to adult, male Sprague-Dawley rats to monitor the route and delay of excreted metabolites in urine and faeces. Peak concentrations appeared in urine after 3.2 1.9 h and in faeces after 16.7 4.3 h. Altogether about 20% of the recovered metabolites were found in urine and about 80% in faeces. Using high-performance liquid chromatography (HPLC ), several peaks of radioacti ve metabolites were found. Some metabolites were detected by enzym e immunoassay (EIA) using two different antibodies (corticosterone, 11b-OH-aet iocholanolone). T here was a marked diurnal variation with low levels of faecal corticosterone metabolites in the evening and higher values in the morning. T his diurnal variation was in¯uenced neither by the intraperitoneal injection of isotonic saline nor by ACT H. However, the administrat ion of dexamethasone eliminated the morning peak for 2 days. Keywords Rat; urine; faeces; corticosterone; ACT H; dexamethasone Adapt at ion to stressful events is associated with an increased production and secretion of glucocorticoids from the adrenal cortex into the blood. Various speci®c and non-speci®c stim uli are able to induce increased secretion of glucocorticoids (Clark e t a l. 1997a,b). When the hypothalam ic-pituit ary-adren al axis is suddenly act ivated, glucocorticoids signi®cantly increase and, in rats, maxim um values are usually seen about 20 min lat er, although the tim e sequence depends on the stim ulus intensity. However, the glucocorticoid concentration in the blood is not an appropriat e indicator of long-term aversive stim ulation, because animals may become less anxious or concerned or may get used to the stim ulus. Also, the feedback regulatory system tends to reduce hormone values (Manser 1992 ). High plasma concentrat ions of glucocorticoids inhibit the release of ACT H from the pituitary gland, which in turn causes a decreased hormone secretion by the adrenal cortex. T herefore, high concentrations of glucocorticoids do not usually persist for a long time (no more than 90 min) in the circulation. Nevertheless, an increase in glucocorticoid concentrations in peripheral blood can represent a sensitive indicator of the intensity of discomfort or distress experienced by the anim als (Van de Kar e t a l. 1991 ).In contrast to the concentration in blood, which is in¯uenced by the stressful sampling itself (Cook e t a l. 1973 ) and which re¯ects a momentary situation, the collection of urine or faeces allows the monitoring of previous

Increased production of glucocorticoids by the adrenal cortex is found in mammals under stress. As cortisol itself is absent in the faeces, an enzyme immunoassay (11-oxoaetiocholanolone) measuring 11,17-dioxoandrostanes has already been established to measure faecal cortisol metabolites in ruminants for non-invasive monitoring of adrenocortical activity. The aim of this study was to establish route and delay of excretion of glucocorticoids in hares and to determine whether a cortisol-, corticosterone- or this new enzyme immunoassay is best suited to detect faecal glucocorticoid metabolites. In the first experiment radioactive-labelled glucocorticoids (14C-cortisol and 3H-corticosterone) were administered intravenously to two groups of three hares in metabolic cages. All voided urine and faecal samples were collected for 4 days. Metabolites of both steroids were found predominantly in the urine (91 +/- 4%). Peak concentrations were observed in the first urinary sample following infusion (13 +/- 6 h) and in the faeces with a delay of about 1 day (23 +/- 7 h). Most of the radioactivity was not extractable with diethylether, indicating that the metabolites excreted in urine and faeces are mainly conjugated or polar unconjugated ones. This was confirmed by reverse-phase high-performance liquid chromatography separations of the metabolites, which also revealed marked differences concerning the metabolism of the two glucocorticoids injected. Compared with the cortisol and the corticosterone enzyme immunoassay, only the group-specific enzyme immunoassay for 11,17-dioxoandrostanes detected high quantities of immunoreactive metabolites. In a second experiment hares (n = 20) were stressed by rousing them three times (5 min, 10 min and another 5 min) with a 20-min break in-between. Faecal samples were collected 2 days before until 4 days after stress and analysed using the 11-oxoaetiocholanolone enzyme immunoassay. After stress significantly (P < 0.001) increased 11,17-dioxoandrostane concentrations were found. Based on these results, measuring 11,17-dioxoandrostanes in faeces enables non-invasive monitoring of disturbances in hares and thus provides a tool for field investigations elucidating the role of stress in hare populations.

Skin biopsies were taken from female dogs, the primary hair follicles isolated and the dermal papilla dissected. After incubation in supplemented Amniomax complete C100 medium in 24-well culture plates, the dermal papilla cells (DPC) grew to confluence within 3 weeks. Thereafter, they were subcultivated every 7 days. Dermal fibroblast (DFB) cultures were established by explant culture of interfollicular dermis in serum-free medium, where they reached confluence in 10 days. They were subcultivated every 5 days. For immunohistochemistry, cells were grown on cover slips for 24 h, fixed and stained with antibodies against collagen IV and laminin. DPC showed an aggregative growth pattern and formation of pseudopapillae. Intensive staining for collagen IV and laminin could be observed until the sixth passage. DFB grew as branching, parallel lines and showed only weak staining for collagen IV and laminin.

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Skin biopsies from seven different body sites were obtained from 21 dogs of different breeds (short, normal, long hair), which were presented for euthanasia. Commercially available polyclonal antibodies were used for the immunohistochemical detection of androgen and oestrogen receptors. Both receptors showed a similar distribution in canine skin, with specific intranuclear staining. In the epidermis, the percentage of androgen receptor (AR)-positive cells, but not that of oestrogen receptor (ER)-positive cells, was significantly higher in samples from the thorax and the flank. In the dermal papilla, the percentage of ER-positive (but not AR-positive) cells was significantly lower in biopsies from the flank. No significant difference was found for both receptors between the locations in the outer root sheath, among the three different hair types, between sex and between intact and castrated dogs.

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ContentsNon-invasive monitoring of the reproductive function in animals is possible by measuring steroid metabolites in the faeces. After infusion of 14C-steroids, differences between species concerning both the percentage and the delay of faecal excretion can be observed. In domestic livestock almost all faecal ''C-metabolites are found in an unconjugated form. The aim of this study was to characterize the main metabolites present in faecal samples of sheep, ponies and pigs after infusion of 14C-progesterone and to evaluate different extraction procedures. This should help to standardize and improve faecal steroid analysis. Faeces containing peak radioactivity were suspended in methanoliwater and Sep-Pak' CI8 cartridges combined with Sephadex LH-20 columns were applied to clean up the samples. Fractions were subjected to straight phase high pressure liquid chromatography (HPLC) and thin layer chromatography (TLC). Metabolites were classified by comparison with the elution patterns of steroid reference standards and by testing their immunoreactivity in different enzyme-immunoassays (EIAs). The results revealed considerable differences in the pattern of faecal progesterone metabolites between species. A great number of metabolites (mainly mono-and di-hydroxylated pregnanes a5 well as pregnanediones) was found and progesterone barely, if at all. In sheep, pregnanes containing a 20a-hydroxy (OH) group dominated (about 45%), whereas in ponies metabolites with a 20fl-OH group (about 44%; mainly one Sa-pregnanediol). and in pigs progestagens with a 20-0x0 group (4658%) predominated. Suspending the faeces with a high percentage of methanol, ethanol or iso-propanol yielded the best recoveries and enabled a practicable extraction, without an evaporation step. As a wide range of metabolites was formed, groupspecific immunoassays using antibodies with high crossreactions with pregnanes sharing a similar C-20 group should be used for measuring faecal metabolites. To verify the data of the 14C-experiments, various progesterone metabolites were measured in faecal samples of superovulated ewes. In accordance with the results of the characterized progesterone metabolites, an EIA measuring 20u-OH-pregnanes proved most suitable for non-invasive monitoring of the corpus luteum function in sheep.