Age‐dependent baseline values of faecal cortisol metabolites in the American mink (Neovison vison) under semi‐natural housing conditions

The welfare of an animal is ensured if it is able to fully satisfy its essential species‐typical needs in all functional aspects of behaviour. In mink, stereotypies and apathy, internal and/or external injuries as well as increased susceptibility to disease have been known to occur as a result of chronic stress. The non‐invasive method of analysing faecal cortisol metabolites (FCM) allows conclusions to be drawn about the stress level in the respective housing system. The objective of this study is to find out how the cortisol metabolites content in the faecal changes with increasing age of the mink under semi‐natural housing conditions. Thus, 40 American mink (Neovison vison) were housed in two outdoor enclosures imitating natural conditions. Throughout the entire study (13th to 32nd week of life), faecal samples were collected to measure cortisol metabolites. No differences in FCM concentrations between the two outdoor enclosures were found. In the young mink lower, less fluctuating FCM levels were found than in older animals. After the first faecal collection in the 13th/14th week of life, the level of metabolites decreased slightly (p = 0.032; 17th/18th week). From the 22nd/23rd week onwards until the 30th/31st week, shortly before the animals were pelted, continuously increasing concentrations were then measured. Increasing FCM levels with advancing age of the animals are probably attributable to the onset of sexual maturity and/or the respective season. This has to be taken into account in future studies using this method for assessing welfare and when comparing different mink housing systems.     

Chondrules with peculiar REE patterns: implications for solar nebular condensation at high C/O

Rare earth element (REE) data from two ordinary chondrite chondrules show distinct negative chondrite-normalized concentration anomalies of samarium, europium, and ytterbium. The peculiar patterns may be the result of REE gas/solid fractionation at an oxygen fugacity lower than has been assumed for the canonical solar nebula. We suggest that the two ordinary chondrite chondrules acquired the fractionated REE patterns by incorporation of highly reduced, ultrarefractory condensates in their precursors. This interpretation implies that high-temperature condensation processes occurred in nebular environments with a strong deficit in oxygen, such as regions with an enhanced carbon/oxygen ratio.     

Hf-W chronometry of lunar metals and the age and early differentiation of the Moon

The use of hafnium-tungsten chronometry to date the Moon is hampered by cosmogenic tungsten-182 production mainly by neutron capture of tantalum-181 at the lunar surface. We report tungsten isotope data for lunar metals, which contain no 181Ta-derived cosmogenic 182W. The data reveal differences in indigenous 182W/184W of lunar mantle reservoirs, indicating crystallization of the lunar magma ocean 4.527 +/- 0.010 billion years ago. This age is consistent with the giant impact hypothesis and defines the completion of the major stage of Earth's accretion.     

Measurement of Faecal Cortisol Metabolites in Cats and Dogs: A Non-invasive Method for Evaluating Adrenocortical Function

The aim of this comparative study was to gain more information about the metabolism and excretion of glucocorticoids in cats and dogs in order to establish non-invasive methods for evaluating stressful conditions. Therefore, in a first experiment, [¹⁴C]cortisol was administered intravenously to 8 animals (two of each sex and species). Over a period of 6 days, faeces and urine were collected immediately after spontaneous defecation and urination. Marked species differences were found, as cats mainly excreted cortisol in the faeces (82%±4% of the total recovered radioactivity), whereas in dogs only a small portion was found there (23%±4%). The highest urinary radioactivity was observed after 9±3 h in cats and 3±1 h in dogs. Peak concentrations in the faeces occurred after 22±6 h in cats and after 24±4 h in dogs. Most of the radioactivity was not extractable with diethyl ether, indicating that the metabolites excreted in urine and faeces were mainly of the conjugated or polar unconjugated types. This was confirmed by RP-HPLC, which also revealed marked differences between cats and dogs concerning the metabolites formed. In addition, the immunoreactivity of the metabolites was tested in cortisol, corticosterone and 11-oxoaetiocholanolone EIAs. The latter, measuring 11,17-dioxoandrostanes (11,17-DOA) detected the highest quantities of immunoreactive metabolites in cats, but not in dogs. In a second experiment, the adrenal cortex of both species was stimulated by ACTH and, three weeks later, suppressed by dexamethasone. In this study, only faeces were collected over a period of 7 days. In both species, inter-animal variability in the basal and maximal/minimal faecal cortisol metabolite concentrations and the time course was observed. The 11-oxoaetiocholanolone EIA in cats and the cortisol EIA in dogs proved best suited for monitoring changes in adrenocortical activity. ACTH injections resulted in an increase above baseline values of 355% (median) in 11,17-DOA concentrations in cats and of 702% in the concentrations of cortisol equivalents in dogs by about 25 h and 22 h (median) after injection, respectively. Minimal concentrations after dexamethasone administration were about 17% in cats and 31% in dogs (in relation to baseline values) and were reached in 66 h and 72 h, respectively. It was concluded that measuring cortisol metabolites in faeces should be a useful non-invasive tool for monitoring stress in carnivores.     

Binding and Clearance of Radioactive Adrenaline and Noradrenaline in Sheep Blood

An understanding of the conditions influencing protein binding of catecholamines (CAs) is important in studying their metabolic effects. Unfortunately, reports on plasma protein binding of CAs are scarce, conflicting and mainly performed in vitro.The aim of our in vivo and in vitro studies was to investigate binding and clearance of radioactive adrenaline (epinephrine) (³H-A), noradrenaline (norepinephrine) (³H-NA) and their metabolites in sheep blood. The time course of the radioactivity in the blood after intravenous injection of ³H-A and ³H-NA (3.7 MBq each) in 4 sheep (2 of each sex; total of 8 administrations) was determined. Blood samples were taken from the jugular vein. The highest radioactivity was observed in the first sample (5 min) following injection. Radioactivity showed a biphasic disappearance. An initial stage, in which radioactivity decreased rapidly (within 1 h) after the injection, was followed by a slow stage, lasting for up to 1 month, until background levels were reached. In vitro results indicated that NA and A were present not only in plasma (70%) but also in the erythrocytes (30%; mainly bound to haemoglobin). Sephadex G-25 gel filtration revealed that from the plasma fraction about 15% was strongly bound to proteins (mainly albumin). These results demonstrate that previous experiments in this field have overestimated the percentage of CAs bound to plasma proteins, because binding to haemoglobin was previously not known. In the future, efforts should be made to characterize the adduct products of CAs and establish an assay to determine them in vivo. If this could be achieved, it would yield a valuable tool for measuring the stress experienced for a longer period.     

Measurement of glucocorticoid metabolite concentrations in faeces of domestic livestock

After 14C-labelled cortisol infusion in ponies and pigs, faecal samples were collected. Extraction of 0.5 g faeces with 5 ml 80-90% methanol yielded the highest radioactivity in the supernatant. Most of the metabolites were ether soluble. After high performance liquid chromatography (HPLC), the presence of immunoreactive metabolites was demonstrated by measuring each HPLC fraction using enzyme immunoassays for cortisol, corticosterone and 11-oxoaetiocholanolone. Only the assay for 11-oxoaetiocholanolone revealed peaks with co-eluting radioactivity. For biological validation of the test system, adrenocorticotrophic hormone (ACTH) and dexamethasone were injected intravenously successively in both species (n = 6). Cortisol concentration in blood and the 11-oxoaetiocholanolone immunoreactive substances in faeces were determined. In horse faeces, basal values of 2.3-35.2 nmol/kg were measured. After ACTH administration, an increase (more than 200% above basal values) of these metabolites was seen about 1 day after ACTH administration. After dexamethasone injection the levels decreased, reaching minimum concentrations 2 days after administration. In pigs, an increase in these metabolites was measured in only three animals after ACTH; dexamethasone did not cause a decrease. The stability of the samples after defecation was tested by storing samples from cows, horses and pigs at room temperature. It was shown that there was a significant increase in the concentration of measured cortisol metabolites in bovine, equine and porcine faeces after storage for 1 h, 4 h and 24 h, respectively. In frozen samples this effect was diminished after thawing samples at 40 degrees C; thawing the samples at 95 degrees C prevented an increase in immunoreactive substances.     

Measurement of Cortisol Metabolites in Faeces of Ruminants

Twenty-one metabolites were detected in faecal samples collected after infusion of (¹⁴C)cortisol into the jugular vein of sheep, using high-performance liquid chromatography/radiometric analysis plus mass spectrometry. One group of metabolites had molecular weights of between 302 and 308, and another group of 350, which indicates that the substances have a C₁₉O₃ or a C₂₁O₄ structure. Therefore, an enzyme immunoassay against 5-androstane-3-o1-11,17-dione-17-CMO:BSA was established. Faecal samples were collected from 10 cows immediately after transport and then during a course in which non-invasive diagnostic procedures were being taught (course 1). For comparison, faeces were sampled from another 5 cows that were being used for teaching invasive procedures (course 2). Six cows from a university farm served as controls. In the animals used in course 1, the highest concentrations of cortisol metabolites were measured immediately after transport to the university (median value: 2.2 mol/kg faeces). During the first 5 days at the university, the concentrations decreased to 0.52 mol/kg (median) and remained at this level during the rest of the course. The median concentration in the samples that were taken during course 2 (collected about 2 months after transport) was 0.48 mol/kg. There was no significant difference in the excretion of cortisol metabolites between these cows and the controls. We conclude from these data that, using the enzyme immunoassay against 5-androstane-3-o1-11,17-dione-17-CMO, we were able to detect transport/novel environment stress but not the potential disturbance that cows experience during diagnostic procedures.     

The measurement of glucocorticoid concentrations in the serum and faeces of captive African elephants (Loxodonta africana) after ACTH stimulation

Conventionally, the assessment of adrenal responses to stress relies on blood sample collection. However, blood collection from animals is impossible without restraint or immobilisation that influences results. This study was undertaken to validate recently established enzyme immunoassays that measure faecal glucocorticoid metabolites in elephants, and to perform a preliminary investigation into the biological relevance of this non-invasive method for use in assessing the degree of stress in this species. Four juvenile African elephants were injected i.m. with 2.15 mg synthetic adrenocorticotrophic hormone (Synacthen, Novartis, Switzerland). Blood and faecal samples were collected over 4 h and 7 d respectively. Concentrations of serum cortisol and faecal cortisol metabolites were determined using immunoassay. Variability of basal and peak values in blood and faeces was observed among the elephants. After ACTH injection, serum cortisol concentrations increased by 400-700%. An 11-oxoaetiocholanolone enzyme immunoassay (EIA) proved best suited to measure cortisol metabolites (11,17-dioxoandrostanes) when compared to a cortisol and corticosterone EIA in faecal samples. Concentrations of faecal 11,17-dioxoandrostanes increased by 570-1070%, reaching peak levels after 20.0-25.5 h. Greater levels of glucocorticoid metabolites were measured in faecal samples from elephants kept in small enclosures compared to levels in the faeces of animals ranging over a larger area. The results of this preliminary study suggest that non-invasive faecal monitoring of glucocorticoid metabolites is useful in investigating adrenal activity in African elephants.