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.

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.

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.

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.

Behavioral and physiological responses of young horses to different weaning protocols: a pilot study.

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.

Measuring corticosterone in seabird egg yolk and the presence of high yolk gestagen concentrations.

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.

Selection for fast and slow exploration affects baseline and stress-induced corticosterone excretion in Great tit nestlings, Parus major.

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.

Changes in cortisol release and heart rate and heart rate variability during the initial training of 3-year-old sport horses.

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.

Cortisol release and heart rate variability in horses during road transport.

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.

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.

Increased yolk testosterone facilitates prenatal perceptual learning in Northern bobwhite quail (Colinus virginianus).

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 hen's 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.

Milk ejection disorders in Swiss dairy cows: a field study.

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.

Effect of giving birth on the cortisol level in a bonobo groups' (Pan paniscus) saliva.

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.

Habituation to humans affects yolk steroid levels and offspring phenotype in quail.

In the field as well as in the laboratory, human-generated stress responses are reduced in adult animals previously habituated to humans in comparison to non-habituated individuals. In birds, yolk steroid levels vary with maternal environment and condition. We tested the hypothesis that the experience of female birds with humans could affect yolk steroids levels and offspring phenotype. Two groups of Japanese quail, one habituated to humans (H) and a second non-habituated (NH), were exposed daily to brief human disturbances. We analysed egg quality, offspring growth, and offspring emotional reactivity. NH females produced eggs with less androgens (testosterone and androstenedione) and more immunoreactive progesterone compared to birds habituated to humans. NH females produced eggs with less yolk, heavier shell and chicks hatching later and being smaller as compared to habituated individuals. A lower emotional reactivity was found in young of NH females compared to young of H females. Thus, human disturbance of the mother triggered different effects on chick phenotype depending on previous experience of mother birds with humans. In addition, we describe for the first time the influence of environmental stimuli on yolk immunoreactive progesterone levels. Our results show that a relatively minor difference in behavioral habituation may have substantial effects on eggs and offspring. This has obvious implications for keeping and handling laboratory animals, for conservation biology and for animal welfare.

Effects of reproductive status and management on cortisol secretion and fertility of oestrous horse mares.

Stressful events may contribute to low reproductive efficiency due to glucocorticoid-mediated inhibition of hormone secretion in a variety of species. We therefore investigated effects of stress related to management of mares around artificial insemination on secretion of cortisol and fertility parameters. To avoid further disturbance of mares by frequent blood sampling, faecal cortisol metabolites (fCM) were determined instead (sample collection at 8-h intervals). A total of 50 mares (16 maiden, 17 barren, 12 foaling, 5 teaching mares) were included in the study. Mares were brought to the AI centre in vans or trailers (driving time between 30 min and 5 h). Teaching mares were housed in the clinic and had therefore not to be transported. Mares were inseminated either with fresh/cooled-shipped or frozen semen. Rectal palpations and ultrasound examinations were performed at 24- to 48-h intervals, in animals inseminated with frozen semen at 6-h intervals during the last 48 h before ovulation. In maiden, barren and foaling mares, fCM concentrations in faeces tended to be higher than in teaching mares at all times after arrival at the AI centre. At 24 and 48 h after arrival, fCM concentrations in maiden mares were significantly higher than in teaching mares (24h: maiden mares 12.3+/-3.1 ng/g, barren mares 8.5+/-1.2 ng/g, foaling mares 11.0+/-2.4 ng/g, teaching mares 3.8+/-0.6 ng/g, p<0.05). The time from arrival at the AI centre to detection of ovulation did not differ among the different groups of mares and was 4.5+/-0.4, 5.0+/-0.5, 3.8+/-0.5 and 5.6+/-0.9 days in maiden, barren, foaling and teaching mares, respectively (n.s.). Pregnancy rates were 53, 53, 55 and 60%, respectively (n.s.). The time from arrival at the AI centre to detection of ovulation was 4.4+/-0.3 days and 4.9+/-0.3 days in mares inseminated with fresh/shipped (n=39) or frozen semen (n=11; n.s.), respectively. The frequency of follicular checks influenced fCM secretion and was statistically significant at 16 h before ovulation (fresh/shipped semen: fCM 6.9+/-0.7 ng/g faeces, frozen semen: fCM 16.9+/-5.2 ng/g faeces, p<0.01). In the mare, gynaecological examinations seem to act as stressors and may increase cortisol secretion. However, this does not negatively influence fertility and in animals familiar with that procedure fCM concentrations are not elevated.

Chronic levonorgestrel treatment in Macaca sylvanus: effects on perineal swelling size and fecal sex steroid excretion.

OBJECTIVE: Administered levonorgestrel enables contraception under normal cyclicity of the ovaries and does not suppress the development of cyclic perineal swellings. This study investigated the influence of levonorgestrel on fecal gonadal steroid excretion rates and the expression of perineal swelling size. SETTINGS: Two groups of Barbary macaque females were observed: Twenty-four implanted individuals under semifree conditions and five non-implanted individuals under caged conditions. Eight of the implanted females had large expressions of the perinea and sixteen reduced. The non-implanted group had no perineal swellings. METHODS: To determine the individual steroid excretion rates and exzymeimmunoassay for decal samples was established. RESULTS: Estradiol excretion rates did not differ in distinctively swollen implanted females, but were increased when compared to non-implanted individuals (df=2; p=0.0002). Implanted females with large perineal swellings had lower progesterone concentrations in the feces compared to individuals with reduced swellings and did not differ from the non-swollen group (df=2; p=0.054). Females with large perineal swellings showed a higher calculated estradiol to progesterone ratio index than the other groups (df=2; p=0.0005). Non-implanted individuals showed increased testosterone excretion rates (df.=2; p=0.0001). MAIN FINDINGS: The results indicate a positive relationship between the perineal swelling size and levonorgestrel implantation. CONCLUSION: The ratio of fecal estradiol and progesterone titers can be judged as an endocrine indicator for the expression rate of perineal swelling size.

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.