Measurement of Fecal Testosterone Metabolites in Mice: Replacement of Invasive Techniques.

Testosterone is the main reproductive hormone in male vertebrates and conventional methods to measure testosterone rely on invasive blood sampling procedures. Here, we aimed to establish a non-invasive alternative by assessing testosterone metabolites (TMs) in fecal and urinary samples in mice. We performed a radiometabolism study to determine the effects of daytime and sex on the metabolism and excretion pattern of radiolabeled TMs. We performed physiological and biological validations of the applied EIA to measure TMs and assessed diurnal fluctuations in TM excretions in male and female mice and across strains. We found that males excreted significantly more radiolabeled TMs via the feces (59%) compared to females (49.5%). TM excretion patterns differed significantly between urinary and fecal samples and were affected by the daytime of ³H-testosterone injection. Overall, TM excretion occurred faster in urinary than fecal samples. Peak excretion of fecal TMs occurred after 8 h when animals received the 3H-testosterone in the morning, or after 4 h when they received the 3H-testosterone injection in the evening. Daytime had no effect on the formed TMs; however, males and females formed different types of TMs. As expected, males showed higher fecal TM levels than females. Males also showed diurnal fluctuations in their TM levels but we found no differences in the TM levels of C57BL/6J and B6D2F1 hybrid males. Finally, we successfully validated our applied EIA (measuring 17ß-hydroxyandrostane) by showing that hCG (human chorionic gonadotropin) administration increased TM levels, whereas castration reduced them. In conclusion, our EIA proved suitable for measuring fecal TMs in mice. Our non-invasive method to assess fecal TMs can be widely used in various research disciplines like animal behavior, reproduction, animal welfare, ecology, conservation, and biomedicine.

A robust high-throughput fungal biosensor assay for the detection of estrogen activity.

Estrogenic active compounds are present in a variety of sources and may alter biological functions in vertebrates. Therefore, it is crucial to develop innovative analytical systems that allow us to screen a broad spectrum of matrices and deliver fast and reliable results. We present the adaptation and validation of a fungal biosensor for the detection of estrogen activity in cow derived samples and tested the clinical applicability for pregnancy diagnosis in 140 mares and 120 cows. As biosensor we used a previously engineered genetically modified strain of the filamentous fungus Aspergillus nidulans, which contains the human estrogen receptor alpha and a reporter construct, in which ß-galactosidase gene expression is controlled by an estrogen-responsive-element. The estrogen response of the fungal biosensor was validated with blood, urine, feces, milk and saliva. All matrices were screened for estrogenic activity prior to and after chemical extraction and the results were compared to an enzyme immunoassay (EIA). The biosensor showed consistent results in milk, urine and feces, which were comparable to those of the EIA. In contrast to the EIA, no sample pre-treatment by chemical extraction was needed. For 17ß-estradiol, the biosensor showed a limit of detection of 1ng/L. The validation of the biosensor for pregnancy diagnosis revealed a specificity of 100% and a sensitivity of more than 97%. In conclusion, we developed and validated a highly robust fungal biosensor for detection of estrogen activity, which is highly sensitive and economic as it allows analyzing in high-throughput formats without the necessity for organic solvents.

Physiological and Behavioral Responses of Dairy Cattle to the Introduction of Robot Scrapers.

Autonomous mobile robot scrapers are increasingly used in order to clean the floors on dairy farms. Given the complexity of robot scraper operation, stress may occur in cows due to unpredictability of the situation. Experiencing stress can impair animal welfare and, in the long term, the health and milk production of the cows. Therefore, this study addressed potential stress responses of dairy cattle to the robot scraper after introducing the autonomous mobile machine. Thirty-six cows in total were studied on three different farms to explore possible modifications in cardiac function, behavior, and adrenocortical activity. The research protocol on each farm consisted of four experimental periods including one baseline measurement without robot scraper operation followed by three test measurements, in which cows interacted with the robotic cleaning system. Interbeat intervals were recorded in order to calculate the heart rate variability (HRV) parameter RMSSD; behavior was observed to determine time budgets; and fecal samples were collected for analysis of the cortisol metabolites concentration. A statistical analysis was carried out using linear mixed-effects models. HRV decline immediately after the introduction of the robot scraper and modified behavior in the subsequent experimental periods indicated a stress response. The cortisol metabolites concentration remained constant. It is hypothesized that after the initial phase of decrease, HRV stabilized through the behavioral adjustments of the cows in the second part of the study. Persistent alterations in behavior gave rise to the assumption that the animals' habituation process to the robot scraper was not yet completed. In summary, the present study illustrated that the cows showed minor signs of disturbance toward the robotic cleaning system. Thus, our findings suggest that dairy cattle can largely adjust their behavior to avoid aversive effects on animal welfare. Additional research can provide further insight into the development of the animal-machine interaction beyond the initial phase of robot scraper operation considered in this study.

Training Reduces Stress in Human-Socialised Wolves to the Same Degree as in Dogs.

The welfare of animals in captivity is of considerable societal concern. A major source of stress, especially for wild animals, is the lack of control over their environment, which includes not being able to avoid contact with human beings. Paradoxically, some studies have shown that interactions with human beings may improve the welfare of wild animals in captivity. Here, we investigated the behavioural (behaviours indicative of cooperation or stress) and physiological (variations in salivary cortisol concentrations) effects of the increasingly used practice of training wild animals as a way to facilitate handling and/or as behavioural enrichment. We evaluated the effects of indoor training sessions with familiar caretakers on nine human-socialised individuals of a wild species, the wolf (Canis lupus), in comparison to nine individuals of its domesticated form, the dog (Canis lupus familiaris). All animals were raised and kept in intraspecific packs under identical conditions-in accordance with the social structure of the species-in order to control for socialisation with human beings and familiarity with training. We also collected saliva samples of trainers to measure GC and testosterone concentrations, to control for the effects of trainers' stress levels on the responses of the animals. During the training sessions, separated from pack members, the animals stayed voluntarily close to the trainers and mostly adequately performed requested behaviours, indicating concentration to the task. Similarly to dogs, the salivary cortisol level of wolves-used as an index of stress-dropped during these sessions, pointing to a similar stress-reducing effect of the training interaction in both subspecies. The responses to the requested behaviours and the reduction in salivary cortisol level of wolves and dogs varied across trainers, which indicates that the relaxing effect of training has a social component. This points to another factor affecting the welfare of animals during the sessions, beside the rewarding effect of getting food and control over the situation by successfully completing a task. As all responses performed by the animals corresponded to cues already familiar to them, the reported effects were likely due to the above cited factors rather than to a learning process. Our results support previous findings suggesting that training is a potentially powerful tool for improving welfare in some wild social canids by creating structured and positive interactions between these animals and their human caretakers.

Sex-specific effects of low-dose gestational estradiol-17ß exposure on bone development in porcine offspring.

Estrogens are important for the bone development and health. Exposure to endocrine disrupting chemicals during the early development has been shown to affect the bone phenotype later in life. Several studies have been performed in rodents, while in larger animals that are important to bridge the gap to humans there is a paucity of data. To this end, the pig as large animal model was used in the present study to assess the influence of gestational estradiol-17ß (E2) exposure on the bone development of the prepubertal and adult offspring. Two low doses (0.05 and 10µg E2/kg body weight) referring to the 'acceptable daily intake' (ADI) and the 'no observed effect level' (NOEL) as stated for humans, and a high-dose (1000µg E2/kg body weight), respectively, were fed to the sows every day from insemination until delivery. In the male prepubertal offspring, the ADI dose group had a lower strength strain index (p=0.002) at the proximal tibia compared to controls, which was determined by peripheral quantitative computed tomography. Prepubertal females were not significantly affected. However, there was a higher cortical cross-sectional area (CSA) (p=0.03) and total CSA (p=0.02) at the femur midpoint in the adult female offspring of the NOEL dose group as measured by computed tomography. These effects were independent from plasma hormone concentrations (leptin, IGF1, estrogens), which remained unaltered. Overall, sex-specific effects on bone development and non-monotonic dose responses were observed. These results substantiate the high sensitivity of developing organisms to exogenous estrogens.

Influence of external factors on hair cortisol concentrations.

Measuring hair cortisol has attracted interest as a long term parameter for chronic stress evaluation. However, some studies support the hypothesis that locally produced cortisol, originating from the hair follicle or skin cells, affects concentrations in the hair. In an animal model the influence of different treatments (extensive brushing, administration of a hyperemising fluid that enhances blood circulation or a synthetic glucocorticoid) on the local cortisol production of hair was evaluated. Therefore eight sheep were sheared and the area of the skin surface of the back was quartered, with three quarters being daily subjected to a certain treatment and one quarter remaining untreated. The skin areas were sheared again after three weeks and cortisol concentrations of all wool samples were determined by immunoassay. Systemic cortisol concentrations were additionally monitored with faecal samples, indicating a significant decline in concentrations of glucocorticoid metabolites between week 1 and 2 or 3, respectively. We found no significant difference in hair cortisol concentrations between fields before treatment (p=0.310). Comparing matched fields before and after treatment, we found no significant differences in wool cortisol concentrations for fields treated with hyperemising fluid as well as for the control fields (p=0.329, p=0.097). Hairs exposed to either extensive brushing or dexamethasone fluid had significantly higher immunoreactive cortisol concentrations after three weeks of treatment (p=0.016, p=0.01). We therefore advise cautious interpretation when measuring hair cortisol concentrations as a parameter for chronic stress, because external factors may have a significant influence on the results.

Cortisol but not testosterone is repeatable and varies with reproductive effort in wild red deer stags.

Although it is known that hormone concentrations vary considerably between individuals within a population, how they change across time and how they relate to an individual's reproductive effort remains poorly quantified in wild animals. Using faecal samples collected from wild red deer stags, we examined sources of variation in faecal cortisol and androgen metabolites, and the potential relationship that these might have with an index of reproductive effort. We also biologically validated an assay for measuring androgen metabolites in red deer faeces. We show that variation in hormone concentrations between samples can be accounted for by the age of the individual and the season when the sample was collected. Faecal cortisol (but not androgen) metabolites also showed significant among-individual variation across the 10-year sampling time period, which accounted for 20% of the trait's phenotypic variance after correcting for the age and season effects. Finally, we show that an index of male reproductive effort (cumulative harem size) during the mating season (rut) was positively correlated with male cortisol concentrations, both among and within individuals. We suggest that the highest ranking males have the largest cumulative harem sizes (i.e. invest the greatest reproductive effort), and that this social dominance may have associated behaviours such as increased frequency of agonistic interactions which are associated with corresponding high levels of faecal cortisol metabolites (FCM).

Immunoreactive cortisone in droppings reflect stress levels, diet and growth rate of gull-billed tern chicks.

Blood levels of corticosterone have been traditionally analyzed to assess stress levels in birds; however, measuring steroid hormone metabolites in feces and droppings has gained much interest as a noninvasive technique successfully used for such purposed in vertebrates. Diet may affect these fecal metabolite levels (e.g., due to nutritional stress), however, this variable has not been taken into account in studies with chicks despite the great dietary flexibility of many avian species. In this study, we addressed for the first time this key issue and validated the technique in wild gull-billed tern chicks (Gelochelidon nilotica). Several enzyme immunoassays were used to determine the most appropriate test to measure the stress response. Subsequently, we performed an experiment in captivity to assess adrenocortical activity in gull-billed tern chicks fed with two diets: piscivorous vs. insectivorous. Finally, the relation between the chicks' growth rate and excreted immunoreactive glucocorticoid metabolites (EGMs) was also evaluated. We found the immunoreactive cortisone metabolites to be a good index of stress (as being an index of adrenocortical reactivity) in chicks of this species. Fish-fed chicks had higher levels of cortisone metabolites when comparing both concentration and total daily excreted metabolites. Within each treatment diet, cortisone metabolite levels and growth rates were negatively correlated. These findings suggest that the diet should be considered when using this technique for comparative purposes and highlight the trade-off between stress levels and chicks growth rates.

Maternally derived egg hormones, antibodies and antimicrobial proteins: common and different pathways of maternal effects in Japanese quail.

Avian eggs contain a variety of maternally-derived substances that can influence the development and performance of offspring. The levels of these egg compounds vary in relation to environmental and genetic factors, but little is known about whether there are correlative links between maternal substances in the egg underlying common and different pathways of maternal effects. In the present study, we investigated genetically determined variability and mutually adjusted deposition of sex hormones (testosterone-T, androstenedione-A4 and progesterone-P4), antibodies (IgY) and antimicrobial proteins (lysozyme) in eggs of Japanese quail (Coturnix japonica). We used different genetic lines that were independently selected for yolk T concentrations, duration of tonic immobility and social reinstatement behaviour, since both selections for behavioural traits (fearfulness and social motivation, respectively) produced considerable correlative responses in yolk androgen levels. A higher selection potential was found for increased rather than decreased yolk T concentrations, suggesting that there is a physiological minimum in egg T levels. Line differences in yolk IgY concentrations were manifested within each selection experiment, but no consistent inter-line pattern between yolk IgY and T was revealed. On the other hand, a consistent inverse inter-line pattern was recorded between yolk IgY and P4 in both selections for behavioural traits. In addition, selections for contrasting fearfulness and social motivation were associated with changes in albumen lysozyme concentrations and an inverse inter-line pattern between the deposition of yolk IgY and albumen lysozyme was found in lines selected for the level of social motivation. Thus, our results demonstrate genetically-driven changes in deposition of yolk T, P4, antibodies and albumen lysozyme in the egg. This genetic variability can partially explain mutually adjusted maternal deposition of sex hormones and immune-competent molecules but the inconsistent pattern of inter-line differences across all selections indicates that there are other underlying mechanisms, which require further studies.

Testing the effect of medical positive reinforcement training on salivary cortisol levels in bonobos and orangutans.

The management of captive animals has been improved by the establishment of positive reinforcement training as a tool to facilitate interactions between caretakers and animals. In great apes, positive reinforcement training has also been used to train individuals to participate in simple medical procedures to monitor physical health. One aim of positive reinforcement training is to establish a relaxed atmosphere for situations that, without training, might be very stressful. This is especially true for simple medical procedures that can require animals to engage in behaviours that are unusual or use unfamiliar medical devices that can be upsetting. Therefore, one cannot exclude the possibility that the training itself is a source of stress. In this study, we explored the effects of medical positive reinforcement training on salivary cortisol in two groups of captive ape species, orangutans and bonobos, which were familiar to this procedure. Furthermore, we successfully biologically validated the salivary cortisol assay, which had already been validated for bonobos, for orangutans. For the biological validation, we found that cortisol levels in orangutan saliva collected during baseline conditions were lower than in samples collected during three periods that were potentially stressful for the animals. However, we did not find significant changes in salivary cortisol during medical positive reinforcement training for either bonobos or orangutans. Therefore, for bonobos and orangutans with previous exposure to medical PRT, the procedure is not stressful. Thus, medical PRT provides a helpful tool for the captive management of the two species.

Ambient temperature and air pressure modulate hormones and behaviour in Greylag geese (Anser anser) and Northern bald ibis (Geronticus eremita).

Ambient temperature and air pressure are relevant stimuli that can elicit hormonal responses in alignment with adjusting individuals' physiology and behaviour. This study investigated possible changes in corticosterone (C) and testosterone (T) and contingencies with behaviour in response to ambient temperature and air pressure, and it evaluated the temporal response dynamics of these hormones in 12 individual Greylag geese (Anser anser) over 26 and 12 individual Northern bald ibis (Geronticus eremita) over 27 days, during late winter. Immunoreactive metabolites of C and T were analysed non-invasively from 626 fecal samples by means of group-specific antibodies and correlated to behaviour and weather factors. In both species, high C levels correlated with low temperatures 24h before sampling, but low C levels correlated with high air pressure 6-12h before sampling. In both species, C levels and behavioural activity were negatively correlated. In addition, temperature had a positive influence on T levels in both species 12-24h before sampling. The fact that weather conditions influenced changes in levels of C, while social interactions did not, is indicative of a general mechanism of graduated physiological adjustment to environmental variations affecting metabolism, stress responses and behaviour.

The role of an alpha animal in changing environmental conditions.

The maintenance and development of conservation areas by grazing of large herbivores, such as Przewalski's horses, is common practice. Several nature conservation areas house male bachelor groups of this species. When males are needed for breeding they are removed from the groups, often without considering group compositions and individual social positions. However, alpha animals are needed for ensuring group stability and decision making in potentially dangerous situations in several species. To investigate the role of the alpha male in a bachelor group, we observed the behaviour of five Przewalski's horse males during the enlargement of their enclosure. We analyzed the group's social structure and movement orders, as well as the animals' connectedness, activity budgets, and whether they moved with preferred group members and how factors such as social rank influenced the horses' behaviour. We also investigated the excretion of glucocorticoid metabolites (GCM) via faeces of the horses while exploring a new area as a parameter of glucocorticoid production. Our results show that the alpha male is important for a bachelor group in changing environmental conditions. The alpha male had the highest level of connectedness within the group. When exploring the new environment, its position in the group changed from previously being the last to being the first. Furthermore the whole group behaviour changed when exploring the new area. The stallions showed reduced resting behavior, increased feeding and did not stay close to each other. We found that the excretion of glucocorticoid metabolites of most horses rose only marginally during the first days on the new area while only the alpha male showed a significant increased amount of glucocorticoid production during the first day of the enclosure enlargement.

Parturition in horses is dominated by parasympathetic activity of the autonomous nervous system.

External and internal stressors prolong parturition in different species. At parturition, sympathoadrenal activation should be avoided because an increased sympathetic tone may cause uterine atonia via ß2-receptors. We hypothesized that at physiological parturition, horses are under parasympathetic dominance, and stress-response mechanisms are not activated during delivery of the foal. To evaluate stress responses, heart rate, heart rate variability, catecholamines, and cortisol were analyzed in mares (n = 17) throughout foaling. Heart rate decreased from 2 hours before (51 ± 1 beats/minute) to 2 hours after delivery (41 ± 2 beats/minute; P < 0.05). Heart rate variability variables, standard deviation of the beat-to-beat interval, and root mean square of successive beat-to-beat differences, changed over time (P < 0.05) with the highest values within 15 minutes after delivery. The number of mares with atrioventricular blocks and the number of atrioventricular blocks per mare increased over time (P < 0.01) and were significantly elevated from 15 minutes before to 45 minutes after birth of the foal. Salivary cortisol concentrations increased to a maximum at 30 minutes after delivery (25.0 ± 3.4 ng/mL; P < 0.01). Plasma epinephrine and norepinephrine concentrations showed significant fluctuations from rupture of the allantochorion to expulsion of the fetal membranes (P < 0.01) but were not markedly elevated at any time. In conclusion, mares give birth under high parasympathetic tone. Cortisol release during and after foaling is most likely part of the endocrine pathways regulating parturition and not a labor-associated stress response.

Measurement of cortisol in dog hair: a noninvasive tool for the diagnosis of hypercortisolism.

BACKGROUND: The clinical signs of hyperadrenocorticism (hypercortisolism) in dogs are known to be caused by chronic overexposure to glucocorticoids. The quantification of cortisol in serum, saliva or urine reflects the cortisol concentration at the time of sample collection, but in suspected hyperadrenocorticism it may be preferable to examine a long-term parameter of cortisol production. HYPOTHESIS/OBJECTIVES: There is a need for a noninvasive method to monitor the long-term production of cortisol in dogs. It seems possible that measuring cortisol levels in hair could represent such a method. ANIMALS: Hair was collected from 12 dogs with hyperadrenocorticism and from 10 healthy control dogs. METHODS: Immunoreactive cortisol, cortisone and corticosterone concentrations were determined by enzyme immunoassay. High-performance liquid chromatography was performed to test the validity of the cortisol assay. RESULTS: Levels of immunoreactive cortisol, cortisone and corticosterone were significantly higher in dogs with hyperadrenocorticism than in control dogs. The difference was most pronounced for the cortisol level. CONCLUSIONS AND CLINICAL IMPORTANCE: The determination of cortisol in hair offers the advantage that sampling is easier and less invasive than taking blood, urine, faeces or saliva. Measuring cortisol in hair may represent a valuable tool for the diagnosis of hyperadrenocorticism in dogs.

Measurements of salivary alpha amylase and salivary cortisol in hominoid primates reveal within-species consistency and between-species differences.

Salivary alpha amylase (sAA) is the most abundant enzyme in saliva. Studies in humans found variation in enzymatic activity of sAA across populations that could be linked to the copy number of loci for salivary amylase (AMY1), which was seen as an adaptive response to the intake of dietary starch. In addition to diet dependent variation, differences in sAA activity have been related to social stress. In a previous study, we found evidence for stress-induced variation in sAA activity in the bonobos, a hominoid primate that is closely related to humans. In this study, we explored patterns of variation in sAA activity in bonobos and three other hominoid primates, chimpanzee, gorilla, and orangutan to (a) examine if within-species differences in sAA activity found in bonobos are characteristic for hominoids and (b) assess the extent of variation in sAA activity between different species. The results revealed species-differences in sAA activity with gorillas and orangutans having higher basal sAA activity when compared to Pan. To assess the impact of stress, sAA values were related to cortisol levels measured in the same saliva samples. Gorillas and orangutans had low salivary cortisol concentrations and the highest cortisol concentration was found in samples from male bonobos, the group that also showed the highest sAA activity. Considering published information, the differences in sAA activity correspond with differences in AMY1 copy numbers and match with general features of natural diet. Studies on sAA activity have the potential to complement molecular studies and may contribute to research on feeding ecology and nutrition.

Moderate heat challenge increased yolk steroid hormones and shaped offspring growth and behavior in chickens.

BACKGROUND: Environmental challenges might affect the maternal organism and indirectly affect the later ontogeny of the progeny. We investigated the cross-generation impact of a moderate heat challenge in chickens. We hypothesized that a warm temperature--within the thermotolerance range--would affect the hormonal environment provided to embryos by mothers, and in turn, affect the morphology and behavioral phenotype of offspring. METHODOLOGY/PRINCIPAL FINDINGS: Laying hens were raised under a standard thermal condition at 21°C (controls) or 30°C (experimental) for 5 consecutive weeks. A significant increase was observed in the internal temperature of hens exposed to the warm treatment; however plasma corticosterone levels remained unaffected. The laying rate was not affected, but experimental hens laid lighter eggs than the controls during the treatment. As expected, the maternal thermal environment affected yolk hormone contents. Eggs laid by the experimental hens showed significantly higher concentrations of yolk progesterone, testosterone, and estradiol. All chicks were raised under standard thermal conditions. The quality of hatchlings, growth, feeding behavior and emotional reactivity of chicks were analyzed. Offspring of experimental hens (C30 chicks) were lighter but obtained better morphological quality scores at hatching than the controls (C21 chicks). C30 chicks expressed lesser distress calls when exposed to a novel food. Unlike C21 chicks, C30 chicks expressed no preference for energetic food. CONCLUSION/SIGNIFICANCE: Our findings suggest that moderate heat challenge triggers maternal effects and modulate the developmental trajectory of offspring in a way that may be adaptive. This suggests that the impact of heat challenges on captive or wild populations might have a cross-generation effect.

Cortisol release, heart rate and heart rate variability in the horse and its rider: different responses to training and performance.

Although some information exists on the stress response of horses in equestrian sports, the horse-rider team is much less well understood. In this study, salivary cortisol concentrations, heart rate (HR) and heart rate variability (HRV), SDRR (standard deviation of beat-to-beat interval) and RMSSD (root mean square of successive beat-to-beat intervals) were analysed in horses and their riders (n=6 each) at a public performance and an identical rehearsal that was not open to the public. Cortisol concentrations increased in both horses and riders (P<0.001) but did not differ between performance and rehearsal. HR in horses and riders increased during the rehearsal and the public performance (P<0.001) but the increase in HR was more pronounced (P<0.01) in riders than in their horses during the public performance (from 91 ± 10 to 150 ± 15 beats/min) compared to the rehearsal (from 94 ± 10 to 118 ± 12 beats/min). The SDRR decreased significantly during the equestrian tasks in riders (P<0.001), but not in their horses. The RMSSD decreased in horses and riders (P<0.001) during rehearsal and performance, indicating a decrease in parasympathetic tone. The decrease in RMSSD in the riders was more pronounced (P<0.05) during the performance (from 32.6 ± 6.6 to 3.8 ± 0.3 ms) than during the rehearsal (from 27.5 ± 4.2 to 6.6 ± 0.6 ms). The study has shown that the presence of spectators caused more pronounced changes in cardiac activity in the riders than it did in their horses.

Assessment of adrenocortical activity by non-invasive measurement of faecal cortisol metabolites in dromedary camels (Camelus dromedarius).

The aim of this study was to determine whether glucocorticoid production could be monitored non-invasively in dromedary camels by measuring faecal cortisol metabolites (FCMs). Five Sudanese dromedaries, two males and three females, were injected with a synthetic adrenocorticotropic hormone (ACTH) analogue. Blood samples were collected pre- and post-ACTH injection. Faeces were sampled after spontaneous defecation for five consecutive days (2 days before and 3 days after ACTH injection). Baseline plasma cortisol values ranged from 0.6 to 10.8 ng/ml in males and from 1.1 to 16.6 ng/ml in females, while peak values after ACTH injection were 10.9-41.9 in males and 10-42.2 ng/ml in females. Peak blood cortisol values were reached between 1.5 and 2.0 h after ACTH injection. The concentration of FCMs increased after ACTH injection in the faeces of both sexes, although steroid levels peaked earlier in males [24 h; (286.7-2,559.7 ng/g faeces)] than in females [36-48 h; (1,182.6-5,169.1 ng/g faeces)], reflecting increases of 3.1-8.3- and 4.3-8-fold above baseline levels. To detect chromatographic patterns of immunoreactive FCMs, faecal samples with high FCM concentrations from both sexes were pooled and subjected to reverse phase high performance liquid chromatography (RP-HPLC). RP-HPLC analysis revealed sex differences in the polarity of FCMs, with females showing more polar FCMs than males. We concluded that stimulation of adrenocortical activity by ACTH injection resulted in a measurable increase in blood cortisol that was reliably paralleled by increases in FCM levels. Thus, measurement of FCMs is a powerful tool for monitoring the adrenocortical responses of dromedaries to stressors in field conditions.

Increased immunoreactive 11-ketotestosterone concentrations in sheep feces after acth challenge.

11-Oxoetiocholanolone and related substances are important metabolites of cortisol and are excreted via feces in ruminants. To investigate whether 11-ketotestosterone (11-KT) or its immunoreactive metabolites are formed and excreted in ruminant feces, an enzyme immunoassay (EIA) was developed and validated. The antibody was raised in rabbits against 11-KT-3-CMO:bovine serum albumin with biotinylated 11-KT as a label. The assay showed a sensitivity of 0.3 pg/well. To validate the assay biologically, 6 rams were injected with a synthetic analogue of the adrenocorticotropic hormone (Synacthen, 2 µg/kg body wt). An aliquot was collected of each fecal portion spontaneously defecated 8 h before Synacthen injection to 24 h after injection and stored at -20 °C until analysis. Samples (0.5 g) were extracted using 80% methanol and immunoreactive metabolites measured using the 11-KT EIA and an already established 11,17-dioxoandrostane (11,17-DOA) EIA. High-performance liquid chromatography separation revealed no peak in the same elution position as authentic 11-KT; therefore, reacting substances were referred to as 11-KT equivalents. In the case of 11-KT immunoreactive substances, the values increased from baseline (median, 136 ng/g feces) to a peak concentration (median, 424 ng/g) 10 to 14 h after Synacthen injection and declined afterwards. Concentrations of 11,17-DOA showed the same pattern, but the values were 2 to 4 times higher. From this data, the authors conclude that 11-KT-like substances, specifically C19 O3 -androgens with a 17ß-hydroxy group, were present in the feces. These substances originate from the adrenals and are most likely cortisol metabolites.

Trans-generational effects of prenatal stress in quail.

The prenatal environment is a source of phenotypic variability influencing the animal's characteristics. Prenatal stress affects not only the development of offspring, but also that of the following generation. Such effects have been best documented in mammals but can also be observed in birds, suggesting common processes across phylogenetic orders. We found previously that Japanese quail females stressed during laying produced offspring with higher fearfulness, probably related to modulation of testosterone levels in their eggs. Here, we evaluated long-term effects of prenatal stress by analysing reproductive traits of these F(1) offspring and, then, the development of their subsequent (F(2)) offspring. The sexual behaviour of F(1) prenatally stressed (F1PS) males was impaired. F1PS females' eggs contained less yolk and more albumen, and higher yolk testosterone and progesterone levels than did F(1) prenatal control females. The fearfulness of F(2) prenatally stressed quail was greater than that of F(2) prenatal control quail. These F(2) behavioural differences paralleled those evidenced by their parents, suggesting trans-generational transmission of prenatal stress effects, probably mediated by egg compositions of F1PS females.