O. Tallo-Parra

Hair cortisol detection in dairy cattle by using EIA: protocol validation and correlation with faecal cortisol metabolites

Hair may be a useful matrix to detect cumulative cortisol concentrations in studies of animal welfare and chronic stress. The aim of this study was to validate a protocol for cortisol detection in hair from dairy cattle by enzyme immunoassay (EIA). Seventeen adult Holstein-Friesian dairy cows were used during the milking period. Hair cortisol concentration was assessed in 25-day-old hair samples taken from the frontal region of the head, analysing black and white coloured hair separately. Concentrations of cortisol metabolites were determined in faeces collected twice a week during the same period of time. There was a high correlation between cortisol values in faeces and cortisol in white colour hair samples but such correlation was not significant with the black colour hair samples. The intra- and inter-assay coefficients of variation were 4.9% and 10.6%, respectively. The linearity showed R 2=0.98 and mean percentage error of -10.8 ± 1.55%. The extraction efficiency was 89.0 ± 23.52% and the parallelism test showed similar slopes. Cortisol detection in hair by using EIA seems to be a valid method to represent long-term circulating cortisol levels in dairy cattle.

Aggressive behavior and hair cortisol levels in captive Dorcas gazelles (Gazella dorcas) as animal-based welfare indicators

Ensuring welfare in captive wild animal populations is important not only for ethical and legal reasons, but also to maintain healthy individuals and populations. An increased level of social behaviors such as aggression can reduce welfare by causing physical damage and chronic stress to animals. Recently, cortisol in hair has been advanced as a non-invasive indicator to quantify long-lasting stress in many species. The sensitivity of social behavior and hair cortisol concentration was evaluated in several groups of dorcas gazelles (Gazella dorcas). Four different groups of gazelles from three different zoos were observed and the expression of intra-specific affiliative and negative social behaviors was assessed across the different groups. Hair samples were taken from sub-groups of animals and analyzed for cortisol concentrations. Significant differences between groups of dorcas gazelles were found in frequency of negative social behavior and hair cortisol concentration. Despite the low sample size, these two parameters had a positive Spearman correlation coefficient (rs  = +0.80, P = 0.20). These results suggest that hair cortisol levels are sensitive to differences in the social structure of dorcas gazelles. Zoo Biol. 35:467-473, 2016.

Heat stress has an effect on motility and metabolic activity of rabbit spermatozoa

In the warm months the function of the spermatozoa can be affected by the temperature of the reproductive tract of the female exposed to hyperthermic conditions. The aim of this study was to evaluate the impact of heat stress on sperm parameters in an in vitro model and to determine if there were seasonal effects on sperm heat tolerance. Sperm samples from 32 New Zealand White rabbits were collected in two seasons and incubated at scrotal (32.5°C), body (37°C) or hyperthermic (42°C) temperatures for 3h. Sperm viability and morphology were evaluated using nigrosin-eosin staining. Motility and metabolic activity parameters were determined using computer-assisted sperm analysis and the QBlue cell viability test, respectively. The incubation of spermatozoa at 42°C decreased (P<0.05) the mean values of total motility, curvilinear (VCL) and mean velocity (VAP) as well as the metabolic activity with respect to the incubation at 32.5°C and 37°C. No seasonal effects were observed except for the highest percentages of bent and coiled tails in the cold season, and the highest mean values of VCL, linear velocity and VAP in the warm season (P<0.01). The interaction between in vitro heat stress and season was significant for metabolic activity (P=0.02). Our results suggest that rabbit spermatozoa parameters are largely modified by a short exposure to hyperthermic conditions, in terms of metabolic activity and motility parameters. Thus, a short exposure of spermatozoa to an environment of 42°C in temperature for only 3h may compromise sperm functionality. Additionally, sperm metabolic activity is influenced by season.

Feather corticosterone evaluated by ELISA in broilers: A potential tool to evaluate broiler welfare

The measure of corticosterone (CORT) in feathers has been recently recognized as a valid and easily obtainable measure of chronic glucocorticoids secretion in avian species. This measure provides meaningful interpretations of how individuals respond to environmental perturbations. The growing interest of the public toward animal-food production welfare shows the need for improving and expanding objective tools to evaluate this issue. The present study evaluates whether it is possible to detect CORT in broiler feathers, and thus, assess if it would be a useful measure to study broiler welfare. Twenty-two broilers were randomly selected from an intensive farm. Four to 6 dorsal feathers were collected from each bird, and sex, weight, and morphological aspects of feather status were recorded. We tested the feasibility for detecting CORT in broiler feathers by ELISA, which had never been done before, and an assay validation test was performed. No significant relationships were found between feather CORT concentrations and physiological variables such as sex, weight, and fault bars in broilers. To our knowledge, this is the first study that uses broiler feathers as a matrix that provides a retrospective record of their hypothalamic-pituitary-adrenal activity. Results indicate that ELISA is a valid tool to detect feather CORT levels in broilers.

Acute ACTH-induced elevations of circulating cortisol do not affect hair cortisol concentrations in calves

In recent times, the detection of cortisol in hair is intended to be used as an animal-based indicator for the assessment of chronic stress. However, the relationship between the ability to report average values of long-term circulating cortisol concentrations and the sensitivity to acute peaks of cortisol is still unclear. To gain insight into this relationship, 24 Holstein-Friesian bull calves under the same management conditions were used in this study. Two injections of ACTH (at D0 and D7) were administrated to twelve animals with the aim to create two acute increases of serum cortisol concentrations. Blood samples were taken in order to determine the duration of serum cortisol peaks and to confirm a mediated response by the administration of ACTH. Cortisol concentrations from 14-day-old white hair samples collected from forehead and hip were analysed separately by EIA and compared with those from the control group. Serum cortisol analyses revealed an acute increase of cortisol concentrations for approximately three hours after each ACTH administration. Concentrations of hair cortisol from forehead and hip locations showed no differences between ACTH-administrated and control animals. Hair cortisol concentrations from 14-day old samples were not altered by two acute elevations of serum cortisol suggesting that hair cortisol is not masked by short and non-recurrent moments of stress. These results are a step forward in the validation of hair cortisol detection as a robust integrative measure of serum cortisol concentrations from an extended period of time.

Relationship between feather corticosterone and subsequent health status and survival in wild Eurasian Sparrowhawk

AbstractIn birds, integrated levels of corticosterone (CORT) measured in feathers (CORTf) allow us to make inferences on past levels of stress demands. It has been suggested that levels of CORTf track carry-over effects across seasons. Nevertheless, our understanding of how this measure can be used to assess future health status is far from complete. In this study, we aimed to investigate whether CORT deposited in feathers over the moulting period was related to subsequent mortality and health status in wild raptors admitted to rehabilitation centres. Thirty-four Eurasian Sparrowhawks (Accipiter nisus) admitted during the non-moulting period were sampled. Body condition (BC) was used as an indicator of health status to classify individuals’ health as good, poor or cachexia depending on their pectoral muscle score. Mortality was recorded over the non-moulting period. Other potential sources of CORTf variation were assessed, such as sex, age and feather type, primary or body covert feathers. While CORTf did not vary with age or sex, significant differences were found between primary and body feathers, highlighting the importance of sampling the same feather type. Our results also revealed that birds in poor BC showed higher CORTf levels than individuals in good condition; however, CORTf levels in cachectic birds did not differ from those in good condition. This finding suggests caution when assuming that only high CORTf levels represent individuals in poor condition, and limits the utility of CORTf for the prediction of BC. The present study also showed that individuals which died following admission had higher CORTf levels than individuals which survived, suggesting the potential utility of CORTf as a metric for the study of subsequent mortality.ZusammenfassungZusammenhang zwischen Feder-Kortikosteron und späterem Gesundheitszustand und Überleben bei Europäischen Sperbern Der Messung von Kortikosteron in Federn (CORTf) erlaubt Rückschlüsse auf vergangene Stresssituationen. Es werde angenommen, dass über CORTf-Spiegel Carry-over Effekte über die Zeit hinweg verfolgt werden können. Gleichwohl ist unser Verständnis, wie solche Werte zur Abschätzung des zukünftigen Gesundheitszustandes genutzt werden können bei Weitem nicht vollständig. Ziel dieser Studie war zu untersuchen, ob in Federn zur Mauserzeit eingelagertes Kortikosteron (CORT) mit späterer Mortalität und dem Gesundheitszustand von Sperbern, die in Greifvogelauffangstationen eingeliefert wurden, zusammenhängen. Dazu beprobten wir 34 außerhalb der Mauserperiode eingelieferte Europäische Sperber (Accipiter nisus). In Abhängigkeit vom Brustmuskelwert wurde die individuelle Gesundheit als gut, schwach oder abgemagert klassifizieren. Zudem wurde die Sterblichkeit über die Nichtmauserzeit ermittelt. Andere potentielle Quellen für Unterschiede im CORTf wie bspw. Geschlecht, Altern und Federtypen, Großgefieder oder Körperfedern wurden ebenfalls berücksichtigt. Während CORTf nicht mit Alter und Geschlecht variierte, wurden signifikante Unterschiede zwischen Groß- und Körpergefieder gefunden, was die Bedeutung der Beprobung desselben Federtyps hervorhebt. Unsere Ergebnisse zeigen auch, dass Vögel mit schlechter Kondition höhere CORTf Werte zeigten als Individuen in guter Kondition, wobei aber die CORTf Werte abgemagerter Vögel sich nicht von denen in guter Kondition unterschieden. Dieses Ergebnis unterstreicht die nötige Vorsicht, wenn angenommen wird, dass nur hohe CORtf Werte für Individuen mit schwacher Kondition stehen und reduziert gleichzeitig die Nützlichkeit von CORTf zur Vorhersage von Körperkonditionen. Die vorliegende Studie zeigt auch, dass Individuen, die später starben, höhere CORTf Werte hatten als Individuen, die überlebten, was zeigt, dass Federkortikosteron als ein Maß für spätere Mortalität verwendet werden kann.

Prediction of Cortisol and Progesterone Concentrations in Cow Hair Using Near-Infrared Reflectance Spectroscopy (NIRS):

Concentrations of different steroid hormones have been used in cows as a measure of adrenal or gonadal activity and, thus, as indicators of stress or reproductive state. Detecting cortisol and progesterone in cow hair provides a long-term integrative value of retrospective adrenal or gonadal/placental activity, respectively. Current techniques for steroid detection require a hormone-extraction procedure that involves time, several types of equipment, management of reagents, and some assay procedures (which can also be time-consuming and can destroy the samples). In contrast, near-infrared reflectance spectroscopy (NIRS) is a multi-component predictor technique, characterized as rapid, nondestructive for the sample, and reagent-free. However, as a predictor technique, NIRS needs to be calibrated and validated for each matrix, hormone, and species. The main objective of this study was to evaluate the predictive value of the NIRS technique for hair cortisol and progesterone quantification in cows by using specific enzyme immunoassay as a reference method. Hair samples from 52 adult Friesian lactating cows from a commercial dairy farm were used. Reflectance spectra of hair samples were determined with a NIR reflectance spectrophotometer before and after trimming them. Although similar results were obtained, a slightly better relationship between the reference data and NIRS predicted values was found using trimmed samples. Near infrared reflectance spectroscopy demonstrated its ability to predict cortisol and progesterone concentrations with certain accuracy (R2 = 0.90 for cortisol and R2 = 0.87 for progesterone). Although NIRS is far from being a complete alternative to current methodologies, the proposed equations can offer screening capability. Considering the advantages of both fields, our results open the possibility for future work on the combination of hair steroid measurement and NIRS methodology.

Metabolic activity of sperm cells: correlation with sperm cell concentration, viability and motility in the rabbit.

The resazurin reduction test (RRT) is a useful technique to assess the metabolic rate of sperm cells. RRT depends on the ability of metabolically active cells to reduce the non-fluorescent dye resazurin to the fluorescent resorufin. The aim of this study was to develop a vital fluorometric method to evaluate metabolic activity of rabbit sperm cells. Twenty-five rabbit males were included in the study. Viability and morphology, motility and metabolic activity were evaluated using an eosin-nigrosin staining, a computer-assisted semen analysis (CASA) and the RRT, respectively. Spearman rank correlation analysis was used to determine the correlation between RRT and semen parameters. After evaluation, a concentration of 10 × 106 sperm cells/ml was selected for further experiments with RRT. No significant correlation was found between the RRT results and the motility parameters. However, after RRT a significant positive correlation between relative fluorescence units and the percentage of alive spermatozoa (r = 0.62; P = 0.001) and a negative one with the percentage of sperm cells with acrosomic abnormalities (r = -0.45; P < 0.05) were detected. The vital assessment of metabolic rate of sperm cells by RRT could provide more information about semen quality than other routine semen analysis, correlating with sperm viability and acrosome status information.

Cortisol detection in fish scales by enzyme immunoassay: Biochemical and methodological validation

The study of fish stress is usually assessed by measuring blood cortisol. Nevertheless, blood provides only a snapshot of the hormonal profile at one point in time. An alternative source of cortisol may be found in scales, providing a new approach for assessing long-term hormonal levels. The present study aimed to develop and validate a methodology for detecting cortisol in scales of goldfish (Carassius auratus). The study highlights the importance of an initial isopropanol washing procedure to completely eliminate external contaminations of cortisol. Additionally, the biochemical validation of the enzyme immunoassay verifies the ability to detect cortisol with repeatability and reliability in goldfish scales. In conclusion, this study provides validated information about a new methodology to measure cortisol in scales. The incorporation of this biomarker could provide retrospective hormonal measurements from species and time periods that are usually difficult or impossible to obtain, thus offering key data of an animal’s physiology. Journal of Applied Ichthyology

Daily salivary cortisol levels in response to stress factors in captive common bottlenose dolphins (Tursiops truncatus): a potential welfare indicator

DOLPHINS in a captive setting can be occasionally subjected to a variety of potentially stressful situations. The stress response comprises a variety of biological reactions to internal or external stimuli elicited when an individual perceives (real or not) a threat to its homoeostasis (Moberg and Mench 2000). The stress response is a complex interplay of behavioural and physiological strategies to cope with changes in the environment (Tsigos and Chrousos 2002, Sheriff and others 2011). Noise can act as a stressor to dolphins because cetaceans rely heavily on sound for many important life functions. This reliance on sound means it is quite likely that exposure to noise will have some detrimental effects on these life functions (Wright and others 2007). Observed effects of noise on marine mammals include: changes in vocalisations, respiration, swim speed, diving and foraging behaviour; displacement; avoidance; shifts in migration path; stress; hearing damage and strandings, but responses of marine mammals to noise can often be subtle and barely detectable (Weilgart 2007). If there is no obvious change in behaviour, stress is difficult to assess in dolphins, both individually and as a group. In order to detect the presence of stress responses in these situations, the endocrine response to stressors is assessed evaluating the hypothalamic–pituitary–adrenal (HPA) axis activity in dolphins and other cetaceans (Wright and others 2007, Amaral 2010). The HPA axis is a key element of the stress response and, when activated, the resultant hormonal cascade increases the levels of glucocorticoids (GCs) …