Gema Silván

Stress physiological responses to tourist pressure in a wild population of European pine marten

The tourist pressure in natural parks is a potential source of stress and may cause an increase in the adrenal activity of wild populations of European pine marten (Martes martes). Seventy-six faecal samples were collected during 15 months in a natural park of Northwest Spain. Analysis of faecal DNA was used for the specific identification using the PCR-RFLPs technique. Faecal steroid determinations were performed by EIA. Natural park was divided in three areas: free entry, restricted area, and integral reservation, and number of daily human visitors recorded. Faecal glucocorticoid metabolite levels (ng/g dry faeces) were significantly higher in spring (56.36+/-19.62) and summer (31.27+/-11.98) compared to autumn (15.33+/-6.89) and winter (11.13+/-3.30). These data are closely related to daily number of visitors (spring: 3204, summer: 1672, winter: 646, autumn: 551). Androgen, progestin and oestrogen levels were also significantly higher in spring (reproductive season) showing values of 43.62+/-18.6, 154.31+/-53.50 and 829.62+/-456.1, respectively. Glucocorticoid levels were significantly lower in integral reservation (15.95+/-3.56) compared to restricted (31.4+/-16.30) and free entry areas (41.59+/-12.73), respectively. Wild populations of European pine marten showed stress physiological response induced by the tourist pressure and this response is higher during reproductive season.

Role of steroid hormones and prolactin in canine mammary cancer

In several animal studies, prolactin has been found to be essential for mammary epithelial development, and its administration has been consistently shown to increase the rate of mammary tumours. High levels of steroid hormones have also been suggested to enhance mammary cancer development. The present study investigates the levels of the following hormones in serum and in tissue homogenates in dogs bearing canine mammary tumours: prolactin (PRL), progesterone (P4), dehydroepiandrosterone (DHEA), androstenedione (A4), testosterone (T), 17beta-estradiol (17beta-E2) and estrone sulfate (S04E1). Eighty mammary tumours (40 dysplasias and benign and 40 malignant tumours) from 32 female dogs, and 10 normal mammary glands from eight female dogs without history of mammary tumours, were analysed. Prolactin and steroid hormones in serum and tissue homogenates, were analysed by enzyme immunoassays (EIA) techniques, previously validated for this animal species. Levels of prolactin in tissue homogenates were significantly different between malignant and benign mammary tumours (p<0.01). Serum prolactin concentrations were lower in the control group as compared with the group of dogs with benign tumours and in dogs with malignant tumours (p=0.01). Serum prolactin levels in dogs with benign lesions were not significantly different than those obtained from dogs with malignant tumours. Levels of steroid hormones were significantly higher in malignant tumours compared with the benign tumours and normal mammary glands (p<0.01) both in serum and homogenate determinations. Our results suggest that the canine neoplastic mammary gland could be a source of prolactin. Our hypothesis is that both prolactin and steroid hormones are involved in the growth of canine mammary cancer, and that they might have an autocrine/paracrine role in the maintenance of this disease.

Steroids and receptors in canine mammary cancer

The aims of this study were to investigate the serum and tissue content of androgens and estrogens in canine inflammatory mammary carcinomas (IMC) as well as in non-inflammatory malignant mammary tumors (MMT), and assessed the immunoexpression of estrogen and androgen receptors using immunohistochemistry. Profiles for the androgens dehydroepiandrosterone (DHEA), androstenedione (A4), and testosterone (T), and for the estrogens 17beta estradiol (E2) and estrone-sulphate (SO4E1) were measured both in tissue homogenates and in serum of MMT and IMC by EIA techniques in 42 non-inflammatory malignant mammary tumors (MMT) and in 14 inflammatory mammary carcinomas (IMC), prospectively collected from 56 female dogs. Androgen receptor (AR) and estrogen receptor alpha (ERalpha) and beta (ERbeta) expression was studied using immunohistochemistry (strepavidin-biotin-peroxidase method) in samples of 32 MMT and 14 IMC, and counted by a computer image analyzer. IMC serum and tissue levels of androgens were significantly higher than MMT levels. Tissue content of estrogens was also significantly higher in IMC than in MMT. Serum values of SO4E1 were significantly higher in IMC, but serum levels of E2 were significantly lower in IMC compared to MMT cases. Medium-high androgen receptor intensity was observed in 64.28% of IMC and 40.62% of MMT. No important differences were found between ERalpha expression in IMC (100% negative) and MMT (90% negative). ERbeta and AR were intensely expressed in highly malignant inflammatory mammary carcinoma cells. To our knowledge, this is the first report relative to AR immunohistochemistry in canine mammary cancer and to estrogens or androgens in serum of dogs with benign or malignant mammary tumors.

Cortisol, adrenocorticotropic hormone, serotonin, adrenaline and noradrenaline serum concentrations in relation to disease and stress in the horse

No detailed comparative data are available on the hormonal parameters of horses suffering from a number of diseases. The aim of our study was to measure concentrations of cortisol, adrenocorticotropic hormone (ACTH), serotonin, adrenaline and noradrenaline in horses with various diseases and following surgery, to assess the response of the HPA axis and adrenal medulla. Blood samples were obtained from six groups of horses comprising a total of 119 animals as follows: laminitis, acute abdominal syndrome (AAS), castration surgery, acute diseases, chronic diseases and healthy controls. Serum hormonal concentrations were determined for each group for comparison. Statistically significant differences between all groups and controls were found for cortisol, ACTH (except for castration), serotonin and adrenaline concentrations but only in horses with laminitis and AAS for noradrenaline. No statistically significant differences were found between males and females. The largest changes in the pituitary-adrenal axis activity occurred mainly in acute diseases, laminitis and in the AAS group.

Crosstalk between GH/IGF-I axis and steroid hormones (progesterone, 17β-estradiol) in canine mammary tumours

Growth hormone (GH), insulin-like growth factor I (IGF-I), progesterone (P4) and 17beta-estradiol (17-E2) concentrations have been studied in 84 mammary tumours (44 dysplasias and benign tumours and 40 malignant neoplasias) from 33 female dogs. Thirteen normal mammary glands from 80 healthy female dogs were also analysed as controls. GH concentrations were determined in mammary homogenates by radio-immunoassay. IGF-I, P4 and 17-E2 tissue levels were determined by enzyme-immunoassay (EIA) techniques. The potential correlations between GH/IGF-I concentrations and P4 and 17-E2 mammary tissue levels were investigated. Tissue GH (p<0.01) and IGF-I concentrations (p<0.01) were significantly higher in malignant tumours than in benign neoplasms. Likewise, malignant tumours were the mammary lesions that displayed the highest P4 and 17-E2 tissue levels. Strong correlations between GH/IGF-I (n=84; r=0.436; p<0.001), P4/GH (n=84; r=0.562; p<0.001) and 17-E2/IGF-I (n=84; r=0.638; p<0.001) were observed in tumoral tissue homogenates. Our study provides evidence that P4 might increase autocrine GH production which might directly stimulate local or systemic IGF-I secretion. Additionally, the IGF-I effect might be influenced by local levels of 17-E2. These results suggest that all these hormones and factors might act as local growth factors stimulating the development and/or maintenance of canine mammary tumours in an autocrine/paracrine manner.

Steroid hormone profile of canine inflammatory mammary carcinoma: a preliminary study

Inflammatory mammary carcinoma (IMC) is the most aggressive spontaneous type of mammary malignant tumor both in women and dogs. Latest studies in dogs indicate that different endocrine mechanisms seem to be involved in inflammatory carcinomas (IMCs). The aim of the present study was to characterize the steroid hormone profile of inflammatory carcinoma, and to compare it with mammary dysplasias, benign tumors and other malignant tumors. Eighty-six mammary samples (10 normal mammary tissue, 21 dysplasias, 26 benign, 22 malignant, and 7 IMC) from 30 female dogs were used. Hormone levels of progesterone (P4), 17beta-estradiol (E2), androstenedione (A4), dehydroepiandrosterone (DHEA), and estrone sulphate (E1SO4) in tissue homogenates were measured by enzyme immunoassays (EIAs) techniques, previously validated for this species. IMC displayed the following steroid profile: P4: 13.80+/-0.56 microg/g; E2: 675.19+/-33.00 ng/g; A4: 631.73+/-70.73 microg/g; DHEA: 702.22+/-89.93 microg/g, and E1SO4: 2.84+/-0.32 mg/g. All of these hormones were significantly higher (P<0.001) compared with the hormone steroid profile determined for malignant, benign, dysplasias, and normal mammary tissue. The most relevant finding was the increased levels, two or three times, of both DHEA and E1SO4 in IMC respect to other groups (P<0.001). These results, together with the highest immunohistochemical expression of P450scc found in IMC, suggest the hypothesis that an autocrine mechanism could be especially involved in the development of canine inflammatory carcinoma.

The effects of different anaesthetic treatments on the adreno-cortical functions and glucose levels in NZW rabbits.

The effects of five anaesthetics on the corticosterone, cortisol and glucose concentrations were investigated in the NZW rabbit. Sixty animals were assigned to 6 treatment groups (n=10 per group): control (iv saline solution injection), ketamine (10 mg/kg iv) with either xylazine (3 mg/kg iv) or diazepam (2 mg/kg iv), pentobarbitone (30 mg/kg iv), thiopentone (20 mg/kg iv) and fentanyl/droperidol (1 mg/kg sc). Plasma glucocorticoids were measured by competitive enzymeimmunoassay EIA and glucose by an autoanalyzer, previously validated for this species in both cases. Blood samples were obtained at 6 time-points: before injection, at 10, 30, 60, 120 min and 24 h after injection of the anaesthetics/saline. A significant decrease of plasma glucocorticoids at 10–60 min was observed in the pentobarbitone and fentanyl/ droperidol groups, whereas the administration of ketamine/diazepam or thiopentone stimulated plasma glucocorticoid release, principally in the recovery period. However, in the ketamine/xylazine group no changes were observed in the glucocorticoid levels, except for a significative increase of cortisol at 60–120 min. Glucose levels significantly increased after ketamine/diazepam administration and principally, after ketamine/xylazine treatment. The present data suggest that ketamine/xylazine has little effect on glucocorticoid levels and provides an adequate level of surgical anaesthesia, hence it would be the anaesthetic of choice, although the hyperglycaemic effect after injection has to be considered for any experimental procedures in rabbits.ResumenSe estudian los efectos de 5 anestésicos sobre las concentraciones de corticosterona, cortisol y glucosa en el conejo NZW. Se utilizan 60 animales divididos en 6 grupos (n=10 por grupo): control (inyección de solución salina iv), fentanil/droperidol (1 mg/kg sc), ketamina (10 mg/kg iv) junto a xilazina (3 mg/kg iv) o diacepán (2 mg/kg iv), pentobarbital (30 mg/kg iv) y tiopental (20 mg/kg iv). Las concentraciones plasmáticas de glucocorticoides se miden mediante la técnica EIA de competición y la glucosa con un autoanalizador. Las muestras de sangre se recogen a los 0, 10, 30, 60, 120 min y 24 horas tras la administración de los anestésicos/solución salina. En los grupos tratados con pentobarbital o fentanil/ droperidol se observa una disminución significativa de los glucocorticoides plasmáticos a los 10–60 min, mientras que la administración de ketamina/diacepán o del tiopental estimula la liberación de glucocorticoides, principalmente durante la recuperación. Sin embargo, tras la administración de ketamina/ xilazina no se observan cambios en los niveles de glucocorticoides, excepto un aumento significativo de cortisol a los 60–120 min. Los niveles de glucosa aumentan significativamente tras la administración de ketamina/diacepán y principalmente, de ketamina/xilazina. Los datos presentes sugieren que la ketamina/xilazina apenas afecta a los niveles de glucocorticoides y alcanza un adecuado nivel de anestesia quirúrgica, por lo que sería el anestésico de elección, aunque el efecto hiperglicémico tras el tratamiento ha de ser considerado en cualquier experimento realizado en conejos.

Relationships Between Sex and Stress Hormone Levels in Feces and Marking Behavior in a Wild Population of Iberian Wolves (Canis lupus signatus)

Feces deposited by the breeding alpha pair on exposed substrates and/or zones may act as visual and olfactory marks associated with social dominance in wolves. The aim of this study was to determine if there was a correlation between marking behavior, sex hormone levels, and physiological stress in a wild population of Iberian wolves in Northwest Spain. The glucocorticoid and sex hormone levels were measured in feces collected as a function of exposure (conspicuous/inconspicuous), height (above ground level/at ground level), and strategic location in the habitat (at crossroads/off crossroads), as well as the frequency of re-marking. The feces, believed to serve as marking cues, had higher glucocorticoid levels (cortisol) and sex hormones (testosterone, progesterone, and estradiol). The results suggest that in Iberian wolves, the alpha pair is subject to higher social stress than subordinate individuals, and that the reproductive suppression of subordinates is not mediated by chronic glucocorticoid elevation.

Effects of tourist pressure and reproduction on physiological stress response in wildcats: Management implications for species conservation

Abstract Context. Ecotourism and human recreational activities are increasing and can have a significant impact on fauna. The analysis of faecal glucocorticoid concentrations is a non-invasive method of measuring physiological stress responses of wildlife to various factors (i.e. human disturbances). Aims. The aim of the present study was to determine whether increased physiological stress levels in wildcats (Felis silvestris) were a response to the level of tourism allowed within different zones in a natural park and/or a response to the seasonal reproductive state of wildcats. Methods. The study was conducted from May 2005 to June 2009 at the Natural Park Montes do Invernadeiro (north-western Spain). The Natural Park is divided into the following three zones according to the level of tourism allowed: restricted public-use, restricted zone and integral reserve. An enzyme immunoassay technique was used to quantify cortisol metabolites and sex hormones from each of 110 fresh wildcat faecal samples collected from walked transects on forest roads within each zone. The number of visitors was recorded as a measure of tourist pressure. Key results. The general linear model indicated that park zone and faecal progesterone levels were the factors that explained the variation in the faecal glucocorticoid metabolite levels. Cortisol metabolite concentrations were higher in some park zones where tourism intensity was higher (restricted public-use zone). Faecal cortisol metabolite concentrations were more elevated during gestation (spring) and during the young dispersal period (autumn). Key conclusions. Therefore, we recommend that some zones of park (integral reserve) continue being maintained free of visitor impact and that visitor numbers be specially controlled during the animals’ sensitive periods (gestation) in the zone of restricted public use and in the restricted zone.

Serum and intratumoural GH and IGF-I concentrations: prognostic factors in the outcome of canine mammary cancer.

The biological implication of the growth hormone/insulin like growth factor-I (GH/IGF-I) axis in canine mammary tumours (CMT) has been recently demonstrated, however its clinical and prognostic implications are unknown. Our aim was to investigate its prognostic significance. Hormonal determinations were done by enzyme immunoassays techniques validated for canine species in serum and tumour tissue from 32 bitches with CMT and in serum and normal mammary tissue from 10 controls. Serum and tissular GH and IGF-I concentrations were significantly higher in the case of malignant tumour compared with benign and controls. GH and IGF-I elevated concentrations were significantly associated with tumour relapse and/or metastases during follow-up and in dogs with reduced survival times; however these parameters were not independent prognostic factors in multivariate analysis. This association demonstrates a link between high serum and intratumoural GH and IGF-I concentrations and a worse prognosis and opens the possibility to new anticancer endocrine therapies in dogs.