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 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.
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 ?2-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.
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.
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.
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.
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.
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.
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.
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.
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.
In this study, effects of weaning on behavioral and physiological stress parameters in young horses (foals) were determined. Foals were weaned either simultaneously without the presence of adult horses (group A, n = 6), or in the presence of two adult females familiar but unrelated to the foals (group B, n = 5), or weaned consecutively by removing two mother horses per day (group C, n = 6). Behavior, locomotion, salivary cortisol concentration, beat-to-beat (RR) interval, heart rate variability (HRV) and weight were determined. Group A foals lost weight for 2 days (mean ± SEM) - 8.3 ± 1.6 kg, p < 0.05. Weaning was followed by increased vocalization which was least pronounced in foals of group B (p < 0.05). Locomotion was most pronounced on weaning day in foals of group A and lowest in group B (p < 0.05). Weaning increased salivary cortisol concentration on the day of weaning in groups A and B and for 2 days in group C (p < 0.05). The RR interval decreased most pronouncedly in group A foals (p < 0.05). There were no consistent changes in HRV. Based on cortisol release and behavior, weaning is associated with stress but this was least pronounced in foals weaned in the presence of two familiar but unrelated adult female horses.
Parents, and particularly mothers, can influence their offsprings development in non-genetic ways. Maternal effects can occur during the mothering phase as well as during the embryonic phase. Prenatal maternal effects in birds can be mediated by yolk steroid hormones that influence subsequent offspring development. Studies have focused mainly on the influence of laying females living conditions on yolk hormonal contents, and rarely on the effects of individual characteristics. Here, we investigated prenatal influence of parent age on yolk steroid levels and on offspring phenotype. We compared Japanese quail at two different ages: at the beginning of their reproductive cycle (11 weeks old: age 1) and six months later, after egg production peak (37 weeks old: age 2). Egg composition, reproductive outcomes, and offspring growth, sexual development and behaviour were studied at both ages. We found that laying rate, fertility and chick survival rates declined between age 1 and age 2. Age 2 eggs had relatively lighter shells and higher yolk plus albumen contents; they also had lower testosterone contents. Age 2 offspring weighed more at hatching than did age 1 offspring; subsequently their growth patterns differed and their sexual development was more precocious. Age 2 offspring were less emotional than age 1 offspring when encountering a novel environment, and they appeared more sensitive to social separation. Our study shows, for the first time in a bird species, a strong impact of parental age on offspring phenotype, and especially on behaviour, an impact that is possibly mediated via modulation of yolk testosterone content.
Salivary alpha-Amylase (sAA) is a starch digesting enzyme. In addition to its function in the context of nutrition, sAA has also turned out to be useful for monitoring sympathetic nervous system activity. Recent studies on humans have found a relationship between intra-individual changes in sAA activity and physical and psychological stress. In studies on primates and other vertebrates, non-invasive monitoring of short-term stress responses is usually based on measurements of cortisol levels, which are indicative of hypothalamic-pituitary-adrenal activity. The few studies that have used both cortisol levels and sAA activity indicate that these two markers may respond differently and independently to different types of stress such that variation in the degree of the activation of different stress response systems might reflect alternative coping mechanisms or individual traits. Here, we present the first data on intra- and inter-individual variation of sAA activity in captive bonobos and compare the results with information from other ape species and humans. Our results indicate that sAA activity in the bonobo samples was significantly lower than in the human samples but within the range of other great ape species. In addition, sAA activity was significantly higher in samples collected at times when subjects had been exposed to stressors (judged by changes in behavioral patterns and cortisol levels) than in samples collected at other times. Our results indicate that bonobos possess functioning sAA and, as in other species, sAA activity is influenced by autonomic nervous system activity. Monitoring sAA activity could therefore be a useful tool for evaluating stress in bonobos.
Reproductive, phenotypic and life-history traits in many animal and plant taxa show geographic variation, indicating spatial variation in selection regimes. Maternal deposition to avian eggs, such as hormones, antibodies and antioxidants, critically affect development of the offspring, with long-lasting effects on the phenotype and fitness. Little is however known about large-scale geographical patterns of variation in maternal deposition to eggs. We studied geographical variation in egg components of a passerine bird, the pied flycatcher (Ficedula hypoleuca), by collecting samples from 16 populations and measuring egg and yolk mass, albumen lysozyme activity, yolk immunoglobulins, yolk androgens and yolk total carotenoids. We found significant variation among populations in most egg components, but ca. 90% of the variation was among individuals within populations. Population however explained 40% of the variation in carotenoid levels. In contrast to our hypothesis, we found geographical trends only in carotenoids, but not in any of the other egg components. Our results thus suggest high within-population variation and leave little scope for local adaptation and genetic differentiation in deposition of different egg components. The role of these maternally-derived resources in evolutionary change should be further investigated.
Large inter-species differences have been found in yolk corticosterone amounts in avian eggs. While some studies have failed to detect significant amounts of corticosterone, in other species high amounts have been recorded, such as in a recent study of southern rockhopper penguins Eudyptes chrysocome chrysocome. However, attention has been drawn recently to the fact that many antibodies for corticosterone measurement cross-react with other steroids present in the yolk. In particular, progesterone and related substances can occur in yolk in high concentrations, such that also low cross-reactions of corticosterone assays may lead to measurement errors. We thus performed high-performance liquid chromatographic (HPLC) analyses of yolk extracts and determined the concentration of immunoreactive corticosterone, as well as cross-reacting progesterone and cortisol in egg yolks of southern rockhopper penguins and imperial shags Phalacrocorax atriceps albiventer. We found that high gestagen concentrations in the yolk result in large measurement errors for yolk corticosterone, even when the cross-reactivity seems small. This was observed for both species. We further found species-specific differences in the actual corticosterone amounts present in the egg yolks.
An animals emotional responses are the result of its cognitive appraisal of a situation. This appraisal is notably influenced by the possibility of an individual to exert control over an aversive event. Although the fact that environment controllability decreases emotional responses in animals is well established, far less is known about its potential trans-generational effects. As the levels of avian yolk hormones can vary according to the mothers environment, we hypothesized that housing environment of mothers would modulate the quality of her eggs and in turn her offsprings behaviour. Two groups of female Japanese quail were constituted: a group that had access to a place to hide in their home-cage (Hd, n?=?20) and a group that had nowhere to hide (NoHd, n?=?20) when stressed. Both groups were submitted to daily human disturbances for a twenty-day-period. Hd females produced eggs with both less testosterone and androstenedione than did NoHd females. The emotional and social reactivity of Hd females offspring were lower and their growth was slower than those of NoHd females offspring. Our results show that a minor difference in housing environment had substantial effects on eggs and offspring. The presence of a shelter probably helped quail to cope with daily human disturbances, producing less reactive offspring. This transgenerational effect caused by an opportunity to hide could lead to applications in care of laboratory animals, conservation biology and animal welfare.
The measurement of faecal glucocorticoid metabolites is used as a non-invasive technique to study stress in animal populations. They have been used most widely in mammals, and mammalian studies have also treated issues such as sample stability and storage methods. In birds, faecal corticosterone metabolite (CM) assays have been validated for a small number of species, and adequate storage under field conditions has not been addressed explicitly in previous studies. Furthermore, while it is well-established that baseline plasma corticosterone levels in birds rise with declining body condition, no study so far investigated if this relationship is also reflected in faecal samples. We here present data of a field study in wild Upland geese Chloephaga picta leucoptera on the Falkland Islands, testing different storage methods and investigating the relationship of faecal CM concentrations to body condition and reproductive parameters. We found that faecal CM measures are significantly repeatable within individuals, higher in individuals with lower body condition in both male and female wild Upland geese and higher in later breeding females with smaller broods. These results suggest that measuring faecal CM values may be a valuable non-invasive tool to monitor the relative condition or health of individuals and populations, especially in areas where there still is intense hunting practice.
Glucocorticoid metabolites enter the aquatic environment via mammalian excrements. Molecular structures of their C19O3 metabolites strongly resemble the major fish androgen 11-ketotestosterone. Therefore, we tested the hypothesis that the cortisol metabolite 5alpha-androstan-3,11,17-trione acts similarly to 11-ketotestosterone by employing a fish screening assay for endocrine-active substances. After 21 d, both 11-oxygenated compounds had masculinized sex characteristics of the anal fin in female medaka in a dose-dependent manner.
Early androgen exposure is known to have long-lasting effects on phenotype, behaviour and even fitness, but difficulties in measuring the exposure hinders the study of its importance in evolutionary context. Digit ratios have been highlighted as a potential easy-to-measure indicator of early steroid exposure, as they have been suggested to reflect steroid, mainly testosterone levels during prenatal development. However, evidence for digit ratios reflecting early steroid levels is weak, as experimental studies, especially in wild populations, are scarce. We studied the association between maternally derived yolk androgens and digit ratios (2D:4D, 2D:3D and 3D:4D) using both correlative data and a rather high level of experimental elevation of yolk androgens in a passerine bird, the pied flycatcher (Ficedula hypoleuca). We also examined whether digit ratios have indicator value in an evolutionary context by studying correlations between digit ratios and reproductive traits, secondary sexual traits and exploratory behaviour. We did not find any association between digit ratios and yolk androgen level either in correlative or experimental data. Digit ratios were neither related to any of the reproductive and secondary sexual traits or exploratory behaviour measured. There was, however, a sex difference in 2D:3D and 3D:4D of adult birds (due to second and fourth digits being shorter in females), which was not apparent in fledglings or captivity-raised juveniles. This suggests that either the sex difference may develop as late as during the sexual maturation for breeding. These results indicate that, in this species, digit ratios are not reliable markers of maternally derived yolk androgen exposure and that they bear little relevance as correlates of the adaptive traits we measured.
Individual phenotypic characteristics of many species are influenced by non-genetic maternal effects. Female birds can influence the development of their offspring before birth via the yolk steroid content of their eggs. We investigated this prenatal maternal effect by analysing the influence of laying females social environment on their eggs hormonal content and on their offsprings development. Social instability was applied to groups of laying Japanese quail females. We evaluated the impact of this procedure on laying females, on yolk steroid levels and on the general development of chicks. Agonistic interactions were more frequent between females kept in an unstable social environment (unstable females) than between females kept in a stable social environment (stable females). Testosterone concentrations were higher in unstable females eggs than in those of stable females. Unstable females chicks hatched later and developed more slowly during their first weeks of life than those of stable females. The emotional reactivity of unstable females chicks was higher than that of stable females chicks. In conclusion, our study showed that social instability applied to laying females affected, in a non-genetic way, their offsprings development, thus stressing the fact that females living conditions during laying can have transgenerational effects.
Female birds might be able to manipulate the parental effort of their male partner through elevated transfer of hormones to the eggs, since these hormones affect many chick traits that males might use as cues for adjusting the level of their investment. We experimentally studied whether female pied flycatchers Ficedula hypoleuca could manipulate male investment via yolk androgens. There is much more variation in yolk androgen levels between females than within clutches, and in order to change the androgen levels of the eggs, we swapped whole clutches between nests. To estimate the androgen levels of the clutch, we measured the androgen content of a single egg per clutch. Females did not succeed in manipulating male effort using yolk androgens, since there was no relationship between the division of parental care within a pair and either original or foster egg androgen levels. One of these relationships should have occurred if females were manipulating males. The proportion of feeding visits by the male was higher when the male was old (55%) than when he was young (45%) and females laid eggs with higher androgen levels when mated with a young male. Young males did not exhibit any responses to yolk androgen levels either, which indicates that females cannot exploit their effort more than that of old males. We suggest that females may allocate yolk androgens to adjust the growth trajectories of the chicks to poor growing conditions when mated with young males that are poor providers or occupying a poor territory.
Based on cortisol release, a variety of situations to which domestic horses are exposed have been classified as stressors but studies on the stress during equestrian training are limited. In the present study, Warmblood stallions (n=9) and mares (n=7) were followed through a 9 respective 12-week initial training program in order to determine potentially stressful training steps. Salivary cortisol concentrations, beat-to-beat (RR) interval and heart rate variability (HRV) were determined. The HRV variables standard deviation of the RR interval (SDRR), RMSSD (root mean square of successive RR differences) and the geometric means standard deviation 1 (SD1) and 2 (SD2) were calculated. Nearly each training unit was associated with an increase in salivary cortisol concentrations (p<0.01). Cortisol release varied between training units and occasionally was more pronounced in mares than in stallions (p<0.05). The RR interval decreased slightly in response to lunging before mounting of the rider. A pronounced decrease occurred when the rider was mounting, but before the horse showed physical activity (p<0.001). The HRV variables SDRR, RMSSD and SD1 decreased in response to training and lowest values were reached during mounting of a rider (p<0.001). Thereafter RR interval and HRV variables increased again. In contrast, SD2 increased with the beginning of lunging (p<0.05) and no changes in response to mounting were detectable. In conclusion, initial training is a stressor for horses. The most pronounced reaction occurred in response to mounting by a rider, a situation resembling a potentially lethal threat under natural conditions.
Glucocorticoids or their metabolites can be measured in several body fluids or excreta, including plasma, saliva, urine and faeces. In recent years the measurement of glucocorticoid metabolites (GCMs) in faeces has gained increasing attention, because of its suitability for wild populations. In horses, however, the group-specific enzyme immunoassay described so far has a limited practicability due to its complex extraction procedure. Therefore, we tested the applicability of other enzyme immunoassays for glucocorticoid metabolites. The present study clearly proved that an enzyme immunoassay (EIA) for 11-oxoaetiocholanolone using 11-oxoaetiocholanolone-17-CMO: BSA (3alpha,11-oxo-A EIA) as antigen showed high amounts of immunoreactive substances. Therefore it was possible to use just a small amount of the supernatant of a methanolic suspension of faeces. The results correlated well with the already described method for measuring GCMs in horse faeces, i.e. analysing the samples with an EIA after a two step clean up procedure of the samples (Merl et al. 2000). In addition, the 3alpha,11-oxo-A EIA has the advantage of providing a bigger difference between baseline values and peak values after ACTH stimulation. The new assay increased the accuracy of the test, lowered the expenses per sample, and storing samples at room temperature after collection was less critical than with other assays investigated in our study. This is a big advantage both in the field of wildlife management of equids and in the field of equestrian sports and it shows the importance of choosing an assay which is in good accordance with the metabolites excreted in a given species.
Public health agencies utilize aerial insecticides to interrupt an active West Nile virus (WNV) transmission cycle, which may expose WNV-infected birds to these agents. Although resmethrin has been considered benign to birds, no studies have evaluated whether the environmentally employed form of resmethrin with PBO synergist (synergized resmethrin (SR)) can suppress avian immunity to WNV infection and enhance a birds host competence. Recognizing that wild birds confront toxicological stressors in the context of various physiological states, we exposed four groups (n=9-11) of 9-week-old chickens (Gallus domesticus) to drinking water with either SR (three alternate days at 50 microg/l resmethrin+150 microg/l piperonyl butoxide), CORT (10 days at 20mg/l to induce subacute stress), the combination of SR and CORT, or 0.10% ethanol vehicle coincident with WNV infection. Compared to controls, SR treatment did not magnify but extended viremia by 1 day, and depressed IgG; CORT treatment elevated (mean, 4.26 log(10)PFU/ml) and extended viremia by 2 days, enhanced IgM and IgG, and increased oral virus. The combination of SR and CORT increased the number of chickens that shed oral virus compared to those treated with CORT alone. None of the chickens developed a readily infectious viremia to mosquitoes (none >or=5 log(10)PFU/ml), but viremia in a CORT-exposed chicken was up to 4.95 log(10)PFU/ml. Given that SR is utilized during WNV outbreaks, continued work toward a complete risk assessment of the potential immunotoxic effects of SR is warranted. This would include parameterization of SR exposures with immunological consequences in wild birds using both replicating (in the laboratory) and non-replicating (in the field) antigens. As a start, this study indicates that SR can alter some immunological parameters, but with limited consequences to primary WNV infection outcome, and that elevated CORT mildly enhances SRs immunotoxicity in chickens.
In nestlings, glucocorticoid (GC) secretion has short-term and long-term fitness consequences. For example, short-time elevations trigger begging activity, whereas chronically elevated GC levels impair body condition, growth and cognitive abilities. Despite a growing body of literature on personality traits, the effects of selection for fast and slow exploration on GC secretion have received little attention. We compared baseline and stress-induced hypothalamic-pituitary-adrenal (HPA) axis activity of hand-reared great tit nestlings of lines selected for fast and slow exploration. Nestling droppings were collected under three conditions: control, test (following handling stress, day 14 after hatching) and the following day. The concentrations of excreted immunoreactive corticosterone metabolites (CM) were determined via an enzyme immunoassay. We also observed nestlings begging behaviour. CM differed significantly between the lines. Nestlings of the fast line excreted lower CM than slow-line birds. In response to handling stress, nestlings excreted significantly higher concentrations of CM than during the control and on the day after handling. Sex and begging activity were not related to CM levels. Under the control condition, but not after handling, males begged significantly more often than females. In both lines, adults excreted significantly less CM compared to nestlings. Both nestlings and adults of the slow line produced higher baseline CM values than fast-line birds. Fast-line nestlings excreted lower baseline CM than nestlings of a wild population not selected for fast or slow exploration. Slow-line nestlings did not. Our results show that selection on the basis of exploratory behaviour affected HPA axis reactivity.
Based on plasma cortisol concentrations it is widely accepted that transport is stressful to horses. So far, cortisol release during transport has not been evaluated in depth by non-invasive techniques such as analysis of salivary cortisol and faecal cortisol metabolites. Transport also causes changes in heart rate and heart rate variability (HRV). In this study, salivary cortisol, faecal cortisol metabolites, heart rate and HRV in horses transported by road for short (one and 3.5 h) and medium duration (8 h) were determined. With the onset of transport, salivary cortisol increased immediately but highest concentrations were measured towards the end of transport (4.1+/-1.6, 4.5+/-2.6, 6.5+/-1.8 ng/ml in horses transported for one, 3.5 and 8 h, respectively). Faecal cortisol metabolite concentrations did not change during transport, but 1 day after transport for 3.5 and 8 h had increased significantly (p<0.01), reflecting intestinal passage time. Compared to salivary cortisol, changes in faecal cortisol metabolites were less pronounced. Heart rate increased and beat-to-beat (RR) interval decreased (p<0.05) with the onset of transport. Standard deviation of heart rate increased while root mean square of successive RR differences (RMSSD) decreased in horses transported for 3.5 (from 74+/-5 to 45+/-6 ms) and 8 h (from 89.7+/-7 to 59+/-7 ms), indicating a reduction in vagal tone. In conclusion, transport of horses over short and medium distances leads to increased cortisol release and changes in heart rate and HRV indicative of stress. The degree of these changes is related to the duration of transport. Salivary cortisol is a sensitive parameter to detect transient changes in cortisol release.
Prenatal learning plays an important role in the ontogeny of behavior and birds provide a useful model to explore whether and how prenatal exposure to hormones of maternal origin can influence prenatal learning and the development of behavior. In this study we assessed if prenatal exposure to yolk testosterone can influence auditory learning in embryos of Northern bobwhite quail (Colinus virginianus). We experimentally enhanced testosterone concentrations in bobwhite quail eggs prior to incubation. The embryos from these T-treated eggs as well as control embryos that had received the vehicle-only or were non-treated were exposed to an individual bobwhite hens maternal call for 120 min over the course of the day prior to hatching. All chicks were tested at 24 h following hatching for their auditory preference between the familiar bobwhite maternal call versus an unfamiliar bobwhite maternal call. T-treated chicks spent significantly more time in proximity to the familiar call compared to the unfamiliar call and also showed shorter latencies to approach the familiar call than control birds. Increased emotional reactivity, i.e. propensity to express fear responses, was also found in T-treated chicks. Baseline heart rates recorded in a second group of T-treated embryos and control embryos did not differ, which suggests no effect of yolk testosterone on baseline arousal level. To our knowledge this is the first demonstration of the influence of prenatal exposure to testosterone on auditory learning.
A questionnaire was sent to 2099 dairy farms to investigate the occurrence of poor milkability. Based on that, the frequency of poor milkability in Swiss dairy cows was 4% and the percentage of cows treated with oxytocin (OT) was 2%. In addition, 270 dairy farms that had reported cases of animals with poor milkability were contacted for an interview to classify the disorders. Farmers suspected disturbed milk ejection in 52%, anatomical dysfunction of the teat and/or the udder in 16% and milk ejection disorder or impaired milkability caused by discernable environmental factors in 32% of the cases. Forty-eight animals from 18 farms with suspected milk ejection disorders were selected for an experimental field study which included milk flow recording and OT administration to induce milk ejection. After cessation of the spontaneous milk flow, a low dose of OT (0.2, 0.5 or 1 i.u.) was injected i.v. to test the responsiveness of the udder to OT at a physiological level. When milk flow ceased again, 10 i.u. OT was injected i.v. (supraphysiological) to ensure complete udder emptying and to determine the residual milk. Milk ejection disorder could be confirmed in 69% of the cases, i.e. if residual milk was >20% of the total milk. Because in 27% of the animals milk ejection disorder was not confirmed on the basis of elevated residual milk, an anatomical disorder of the teat and/or the udder was suspected. Milk ejection disorder could be confirmed in 69% of the cases whereas in 27% of the suspected cases an anatomical disorder of the teat and/or the udder was suspected. An increased cortisol production in cows with milk ejection disorder was not obvious because faecal concentrations of cortisol metabolites with a 5 beta-androstane-3 alpha,11 oxo-structure were not augmented in animals with disturbed milk ejection.
This study documents the cortisol levels in the saliva of a bonobo group, especially that of a bonobo female which had given birth for the first time. During a long study in Zoological Garden Frankfurt, Germany, a bonobo baby was born on 3 August 2007. Due to the fission-fusion keeping system employed, the bonobos were divided into two groups on this day. Their behavior was observed regularly and saliva was also collected. The bonobos had been trained to chew cotton wool and to give back the samples. The cortisol response was tested twice a day before birth and three times on the day of parturition. Before birth, no observable indication behavior was seen, but an increase in the cortisol concentration of the expectant mother was found. Parturition occurred at 8 pm. The next morning, the group with the newborn was visibly more active, which correlated with the fact that their cortisol levels were increased in the morning in comparison to the second group. During the day, cortisol decreased in both groups, only it was higher throughout the day in the new mother. In the evening, the two groups showed nearly the same cortisol levels. These data indicate that there is indeed a relation between observable behavior and the cortisol level in bonobo saliva. Therefore, the cortisol level can be regarded as a suitable indicator for verifying behavioral events.
The prenatal environment is a source of phenotypic variability influencing the animals 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.
Tertiary wastewater treatment plant effluent before and after ozonation (0.6-1.1g O?/g DOC) was tested for aquatic ecotoxicity in a battery of standardised microbioassays with green algae, daphnids, and zebrafish eggs. In addition, unconjugated estrogen and 17?-hydroxyandrogen immunoreactive substances were quantified by means of enzyme immunoassays, and endocrine effects were analysed in a 21-day fish screening assay with adult male and female medaka (Oryzias latipes). Ozonation decreased estrogen-immunoreactivity by 97.7±1.2% and, to a lesser extent, androgen-immunoreactivity by 56.3±16.5%. None of the short-term exposure ecotoxicity tests revealed any adverse effects of the tertiary effluent, neither before nor after the ozonation step. Similarly in the fish screening assay, reproductive fitness parameters showed no effects attributed to micropollutants, and no detrimental effects of the effluents were observed. Based on the presented screening, ozonation effectively reduced steroid hormone levels in the wastewater treatment plant effluent without increasing the effluents ecotoxicity.
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