Addressing Combative Behaviour in Spanish Bulls by Measuring Hormonal Indicators.

The fighting bull is characterised by its natural aggressiveness, but the physiological mechanisms that underlie its aggressive behaviour are poorly studied. This study determines the hormonal component of aggressiveness in fighting bulls by analysing their behaviour during a fight and correlating it to their serotonin, dopamine and testosterone levels. We also determine whether aggressive behaviour can be estimated in calves. Using 195 animals, samples were obtained when the animals were calves and after 5 years. Aggressiveness scores were obtained by an observational method during bullfights, and serotonin, dopamine and testosterone levels were determined in all animals using validated enzyme immunoassay kits. The results revealed a strong correlation of serotonin and dopamine levels with aggressiveness scores in bulls during fights, but no correlation was found with respect to testosterone. These correlations led to established cut-off point and linear regression curves to obtain expected aggressiveness scores for calves at shoeing. There were no significant differences between the expected scores obtained in calves and the observed scores in bulls. Therefore, this study demonstrates that hormone determination in calves may be a great indicator of combativeness in bulls and can reliably be used in the selection of fighting bulls.

Androgen and Estrogen ß Receptor Expression Enhances Efficacy of Antihormonal Treatments in Triple-Negative Breast Cancer Cell Lines.

The triple-negative breast cancer (TNBC) subtype is characterized by the lack of expression of ERα (estrogen receptor α), PR (progesterone receptor) and no overexpression of HER-2. However, TNBC can express the androgen receptor (AR) or estrogen receptor ß (ERß). Also, TNBC secretes steroid hormones and is influenced by hormonal fluctuations, so the steroid inhibition could exert a beneficial effect in TNBC treatment. The aim of this study was to evaluate the effect of dutasteride, anastrozole and ASP9521 in in vitro processes using human TNBC cell lines. For this, immunofluorescence, sensitivity, proliferation and wound healing assays were performed, and hormone concentrations were studied. Results revealed that all TNBC cell lines expressed AR and ERß; the ones that expressed them most intensely were more sensitive to antihormonal treatments. All treatments reduced cell viability, highlighting MDA-MB-453 and SUM-159. Indeed, a decrease in androgen levels was observed in these cell lines, which could relate to a reduction in cell viability. In addition, MCF-7 and SUM-159 increased cell migration under treatments, increasing estrogen levels, which could favor cell migration. Thus, antihormonal treatments could be beneficial for TNBC therapies. This study clarifies the importance of steroid hormones in AR and ERß-positive cell lines of TNBC.

Predicting Sex in White Rhinoceroses: A Statistical Model for Conservation Management.

Ensuring the effective management of every rhinoceros population is crucial for securing a future for the species, especially considering the escalating global threat of poaching and the challenges faced in captive breeding programs for this endangered species. Steroid hormones play pivotal roles in regulating diverse biological processes, making fecal hormonal determinations a valuable non-invasive tool for monitoring adrenal and gonadal endocrinologies and assessing reproductive status, particularly in endangered species. The purpose of this study was to develop a statistical model for predicting the sex of white rhinoceroses using hormonal determinations obtained from a single fecal sample. To achieve this, 562 fecal samples from 15 individuals of the Ceratotherium simum species were collected, and enzyme immunoassays were conducted to determine the concentrations of fecal cortisol, progesterone, estrone, and testosterone metabolites. The biological validation of the method provided an impressive accuracy rate of nearly 80% in predicting the sex of hypothetically unknown white rhinoceroses. Implementing this statistical model for sex identification in white rhinoceroses would yield significant benefits, including a better understanding of the structure and dynamics of wild populations. Additionally, it would enhance conservation management efforts aimed at protecting this endangered species. By utilizing this innovative approach, we can contribute to the preservation and long-term survival of white rhinoceros populations.

Physiological Stress Responses in Cattle Used in the Spanish Rodeo.

Certain events can cause distress in cattle. In Spain, there is a sport similar to rodeo called persecution and takedown, in which calves are harassed and knocked down by riders. In this study, the physiological stress response of calves (n = 260) is assessed by measuring hormonal physiological parameters. Salivary samples were collected from Salers (n = 110) and Lidia (n = 150) calves before, during, and after the persecution and takedown event. The hormones epinephrine, cortisol, serotonin, and dopamine were determined in saliva samples using enzyme-immunoassay techniques. The results obtained revealed that epinephrine and cortisol levels increased during the event in Salers calves, with a significant increase (p < 0.05) in the case of epinephrine, although after the event, these values returned to their initial state. Therefore, this sport supposes an assumable punctual stressor stimulus for the animal. In contrast, in Lidia calves, cortisol and epinephrine levels decreased, with a significant decrease (p < 0.05) in the case of cortisol, which may be related to the temperament of this breed and facing a stressful situation in a different manner. This is confirmed by serotonin and dopamine levels that were altered in Lidia calves with respect to the other group studied. In conclusion, the sport of persecution and takedown produces a physiological response of adaptive stress assumable for the animals.

The inhibition of steroid hormones determines the fate of IPC-366 tumor cells, highlighting the crucial role of androgen production in tumor processes.

Inflammatory mammary cancer (IMC) is a disease that affects female dogs. It is characterized by poor treatment options and no efficient targets. However, anti-androgenic and anti-estrogenic therapies could be effective because IMC has a great endocrine influence, affecting tumor progression. IPC-366 is a triple negative IMC cell line that has been postulated as a useful model to study this disease. Therefore, the aim of this study was to inhibit steroid hormones production at different points of the steroid pathway in order to determine its effect in cell viability and migration in vitro and tumor growth in vivo. For this purpose, Dutasteride (anti-5αReductase), Anastrozole (anti-aromatase) and ASP9521 (anti-17ßHSD) and their combinations have been used. Results revealed that this cell line is positive to estrogen receptor ß (ERß) and androgen receptor (AR) and endocrine therapies reduce cell viability. Our results enforced the hypothesis that estrogens promote cell viability and migration in vitro due to the function of E1SO4 as an estrogen reservoir for E2 production that promotes the IMC cells proliferation. Also, an increase in androgen secretion was associated with a reduction in cell viability. Finally, in vivo assays showed large tumor reduction. Hormone assays determined that high estrogen levels and the reduction of androgen levels promote tumor growth in Balb/SCID IMC mice. In conclusion, estrogen levels reduction may be associated with a good prognosis. Also, activation of AR by increasing androgen production could result in effective therapy for IMC because their anti-proliferative effect.

Long-Term Exposure to Isoflavones Alters the Hormonal Steroid Homeostasis-Impairing Reproductive Function in Adult Male Wistar Rats.

The consumption of isoflavones is gaining popularity worldwide due to their beneficial effects on health. However, isoflavones are considered to be endocrine disruptors and cause deleterious effects on hormone-sensitive organs, especially in males. Therefore, this study aimed to determine if a continuous and prolonged exposure to isoflavones in adult males altered the endocrine axis effect of testicular function. For this purpose, seventy-five adult male rats were administered with low and high mixtures of isoflavones (genistein and daidzein) for 5 months. The determination of steroid hormones (progesterone, androstenedione, dehydroepiandrosterone, testosterone, dihydrotestosterone, 17ß-estradiol, and estrone sulphate) was carried out in serum and testicular homogenate samples. Sperm quality parameters and testicular histology were also determined. The results revealed that low and high doses of isoflavones promote a hormonal imbalance in androgen and estrogen production, resulting in a decrease in circulating and testicular androgen levels and an increase in estrogen levels. These results are associated with a reduction in the sperm quality parameters and a reduction in the testicular weight, both in the diameter of the seminiferous tubules and the height of the germinal epithelium. Altogether, these results suggest that a continuous exposure to isoflavones in adult male rats causes a hormonal imbalance in the testes that disrupts the endocrine axis, causing defects in testicular function.

Hormonal Homologies between Canine Mammary Cancer and Human Breast Cancer in a Series of Cases.

The validity of spontaneous canine mammary cancer (CMC) as a natural model for the study of human breast cancer (HBC) from a hormonal point of view has never been thoroughly investigated. In this study, we analyzed the immunohistochemical expression of aromatase (Arom) and steroid receptors [estrogen receptor α (ER α), estrogen receptor ß (ER ß), progesterone receptor (PR) and androgen receptor (AR)] and intratumor steroid hormone levels of 17ß-estradiol (E2), estrone sulfate (SO4E1), progesterone (P4), androstenedione (A4), dehydroepiandrosterone (DHEA), and testosterone (T) in 78 samples of mammary cancer-51 human breast cancer (HBC) and 27 canine mammary cancer (CMC)-and corresponding controls. Frequency of tumors expressing Arom, ERß, PR, and AR was similar in both species, whereas ERα+ tumors were less frequent in the canine species. There was a closer similarity between premenopausal HBC and CMC. In HBC and CMC, all hormones assayed were increased in tumors compared to control samples. Intratumor androgen levels were similar in the two species, although levels of progesterone and estrogens were higher in the HBC samples than the CMC samples. Statistical associations among Arom, receptors, and hormones analyzed suggest that the major hormonal influence in both species is estrogenic through the ER, being the α isoform predominant in the human samples. Our findings further support CMC as a spontaneous model for the study of HBC, especially premenopausal HBC, although several differences, such as the more prevalent ERα immunoexpression and higher intratumor levels of estrogens and P4 in HBC, should be taken into account in comparative hormonal studies.

Preliminary Findings on How Different Management Systems and Social Interactions Influence Fecal Glucocorticoid Metabolites in White Rhinoceros (Ceratotherium simum).

White rhinoceroses (Ceratotherium simum) are the most social and gregarious species of all rhinoceroses known worldwide. One of the most critical effects of elevated glucocorticoid concentrations, especially in threatened species, is its relation to chronic stress, which could potentially lead to immunosuppression and reduced reproductive activity. Our aim is to determine how different social environments and management systems might be influencing the secretion of fecal glucocorticoids in white rhinoceroses. We have analyzed the concentration of fecal glucocorticoid metabolites in 658 fecal samples from 14 white rhinoceroses, seven free-ranging rhinos, and seven rhinos from two different managed captive populations. HPLC techniques were used to determine the main glucocorticoid metabolite found in this species, and a competitive EIA was used to establish fecal 5α-pregnan-3 3ß, 11ß, 21-triol-20-oneglucocorticoid metabolite (fGCM) levels. Our results reveal that management systems and social interactions had an influence on fGCM levels, suggesting that the more restrictive the management and social conditions are, the higher the glucocorticoid concentrations that are found. Additionally, sex was also found to influence fGCM levels, as in females, fGCM concentrations were higher than in males. We conclude that the analysis of glucocorticoids in relation to other factors is a powerful tool to assess adrenocortical response in white rhinoceros in order to broaden the knowledge of their reproductive biology and improve the management of the species.

Blocking Estrogen Synthesis Leads to Different Hormonal Responses in Canine and Human Triple Negative Inflammatory Breast Cancer.

Blocking estrogen synthesis by inhibitors of estrogen synthesis is a widely used therapy against estrogen receptor-positive tumors. However, these therapies are less effective in negative expression tumors. Therefore, this study determined the effectiveness of anti-aromatase and anti-sulfatase therapies in canine and human inflammatory breast cancer. Cell cultures and xenografts from IPC-366 and SUM149 were treated with different doses of letrozole (anti-aromatase) and STX-64 (anti-sulfatase), in order to observe their effectiveness in terms of cell proliferation, tumor progression, and the appearance of metastases and hormonal profiles. The results revealed that both treatments are effective in vitro since they reduce cell proliferation and decrease the secreted estrogen levels. In xenograft mice, while treatment with letrozole reduces tumor progression by 30-40%, STX-64 increases tumor progression by 20%. The hormonal results obtained determined that STX-64 produced an increase in circulating and intratumoral levels of estradiol, which led to an increase in tumor progression. However, letrozole was able to block estrogen synthesis by decreasing the levels of circulating and intratumoral estrogen and thus slowing down tumor progression. In conclusion, letrozole can be an effective treatment for canine and human inflammatory breast cancer. The knowledge of the hormonal profile of breast tumors reflects useful information on the effectiveness of different endocrine treatments.

Non-Invasive Determination of Annual Fecal Cortisol, Androstenedione, and Testosterone Variations in a Herd of Male Asian Elephants (Elephas maximus) and Their Relation to Some Climatic Variables.

The measurement of stress and reproductive hormones in wild animal species by non-invasive methods is of special interest. To assess whether the adrenal and gonadal hormones show annual variations in male Asian elephants (Elephas maximus) and to evaluate whether there is any influence of climatic variables on hormonal secretion, fecal samples were taken from a herd of 7 Asian elephants over a 14-month period to subsequently determine the concentrations of testosterone (T), androstenedione (A4), and cortisol (C) by a validated immunoassay technique. Data referring to three climatic variables in the place and period of study were collected, namely monthly mean values of temperature, humidity and rainfall. Levels of T and A4 showed two major increases in July (T: 1088.35 ± 131.04 ng/g; A4: 480.40 ± 50.86 ng/g) and October (T: 825.09 ± 31.60 ng/g; A4: 319.96 ± 32.69 ng/g) (p < 0.05). Our results show a secretion of fecal androgens dependent on temperature (T and A4), and humidity (T). Male musth was detected during the initial increases of T and A4 levels. The highest concentrations of C were observed in September (156.67 ± 60.89 ng/g) (p < 0.05), probably due to the stressful fights that occurred during the musth period. The observed results of the fecal levels of T, A4 and C were similar to those obtained by invasive methods. In conclusion, fecal secretion of the three hormones in these captive male Asian elephants showed variations related in some cases to different weather factors.

Tumor Growth Progression in Ectopic and Orthotopic Xenografts from Inflammatory Breast Cancer Cell Lines.

Xenografts can grow in immunosuppressed hosts, such as SCID mice, and tumor material can be injected into hosts either ectopically or orthotopically. Choosing the correct model to use is a crucial step in animal research. The aim of this study was to report the differences between ectopic and orthotopic xenografts in tumor progression, metastasis capacity, histological features, and steroid hormone profiles in xenografts from the cIMC (canine inflammatory mammary cancer) cell line IPC-366 and hIBC (human inflammatory breast cancer) cell line SUM149. To achieve this purpose, 40 female mice 6-8 weeks old were inoculated with IPC-366 and SUM149 cells subcutaneously (ectopic models) or into mammary fat pad (orthotopic models). Mice were monitored for tumor progression and appearance of metastases, and generated tumors were analyzed in terms of histological examination and steroid hormone production. The results revealed differences in tumor appearance and percentage of metastasis between ectopic and orthotopic models, which were higher in the ectopic xenografts from both cell lines. However, both models had similar characteristics of tumor progression, histological features, and steroid hormone secretion profiles. We show that the ectopic model can be validated as a good and useful model of tumor development in addition to, not contrary to, the orthotopic model in breast cancer research.

Accurate prediction of birth implementing a statistical model through the determination of steroid hormones in saliva.

Steroidal hormone interaction in pregnancy is crucial for adequate fetal evolution and preparation for childbirth and extrauterine life. Estrone sulphate, estriol, progesterone and cortisol play important roles in the initiation of labour mechanism at the start of contractions and cervical effacement. However, their interaction remains uncertain. Although several studies regarding the hormonal mechanism of labour have been reported, the prediction of date of birth remains a challenge. In this study, we present for the first time machine learning algorithms for the prediction of whether spontaneous labour will occur from week 37 onwards. Estrone sulphate, estriol, progesterone and cortisol were analysed in saliva samples collected from 106 pregnant women since week 34 by enzyme-immunoassay (EIA) techniques. We compared a random forest model with a traditional logistic regression over a dataset constructed with the values observed of these measures. We observed that the results, evaluated in terms of accuracy and area under the curve (AUC) metrics, are sensibly better in the random forest model. For this reason, we consider that machine learning methods contribute in an important way to the obstetric practice.

Longitudinal study on steroid hormone variations during the second trimester of gestation: a useful tool to confirm adequate foetal development.

BACKGROUND: The interaction of hormonal factors are crucial for good foetal development. During the second trimester of gestation, most of the main physiological processes of foetal development occur. Therefore, the aim of this study was to determine the variations in the physiological levels of cortisol, estriol, estrone sulphate, and progesterone during the second trimester (weeks 12-26) in order to establish normal ranges that can serve as indicators of foetal well-being and good functioning of the foetal-placental unit. METHODS: Saliva samples from 106 pregnant women were collected weekly (from week 12 to week 26 of gestation), and hormonal measurements were assayed by an enzyme immunoassay. The technique used for hormone measurements was highly sensitive and served as a non-invasive method for sample collection. RESULTS: The results revealed a statistically significant (p<0.05) difference between cortisol, progesterone, and oestrogens throughout the second trimester, with a more substantial relationship between oestrogens and progesterone [P4-E3 (r=0.427); P4-E1SO4 (r=0.419)]. By analysing these hormone concentrations, statistically significant (p<0.05) elevations in progesterone, cortisol, and estriol levels were found at the 16th [(P4 (0.78±0.088), C(1.99±0.116), E3(2.513±0.114)]; 18th [(P4 (1.116±0.144), C(3.409±0.137), E3(3.043±0.123)] and 23rd week of gestation [(P4(1.36±0.153), C(1.936±0.11), E3(2.657±0.07)]. Estrone sulphate levels appeared to increase progressively throughout the second trimester [from 1.103±0.03 to 2.244±0.09]. CONCLUSION: The 18th week of gestation seems to constitute a very important week during foetal adrenal development, and the analysis of the main hormones involved in foetal development, provided more precise information regarding the proper functioning of the foetal unit and foetal development.

Reproductive hormones monthly variation in free-ranging European wildcats: Lack of association with faecal marking.

Integrating ecophysiological and behavioural discoveries in conservation and management plans is essential to preserve scarce and elusive species such as the European wildcat (Felis silvestris). The purpose of this study was to characterize the monthly variation in the steroid reproductive hormone metabolite levels (oestradiol, progesterone and testosterone) in this species and to test its possible association with a monthly pattern of faecal marking. By collecting fresh faecal samples in Montes do Invernadeiro Natural Park (Galicia, Northwest Spain) each month, we obtained a total of 110 samples belonging to 25 different individuals. We conducted enzyme immunoassays which allowed us to track the annual variation in reproductive hormone excretion patterns in wildcat scats. Furthermore, we also evaluated the possible relation between the faeces used as marks and the reproductive hormone levels. We found that oestradiol and progesterone metabolite levels exhibited a distinct pattern, both increasing during the breeding months. Oestradiol metabolite larger peaks were found during March and April, whereas the highest concentration of progesterone metabolites appeared in July. On the contrary, testosterone metabolite levels did not significantly change depending on the month. Moreover, we did not find any evidence that the faecal marking behaviour pattern was associated with reproductive hormone metabolite levels. It seems that other factors related to habitat and food resources could be more important in the performance of this behaviour.

Effects of doxorubicin associated with amniotic membrane stem cells in the treatment of canine inflammatory breast carcinoma (IPC-366) cells.

BACKGROUND: Tumours in mammary glands represent the most common neoplasia in bitches, as in humans. This high incidence results in part from the stimulation of sex hormones on these glands. Among mammary tumours, inflammatory carcinoma is the most aggressive, presenting a poor prognosis to surgical treatment and chemotherapy. One of the most widely used chemotherapy drugs for breast cancer treatment is doxorubicin (DOXO). Alternative therapies have been introduced in order to assist in these treatments; studies on treatments using stem cells have emerged, since they have anti-inflammatory and immunomodulatory properties. The aim of this study was to evaluate the effects of DOXO and canine amniotic membrane stem cells (AMCs) on the triple-negative canine inflammatory mammary carcinoma cell line IPC-366. METHODS: Four experimental groups were analysed: a control group without treatment; Group I with DOXO, Group II with AMC and Group III with an association of DOXO and AMCs. We performed the MTT assay with DOXO in order to select the best concentration for the experiments. The growth curve was performed with all groups (I-III) in order to verify the potential of treatments to reduce the growth of IPC-366. For the cell cycle, all groups (I-III) were tested using propidium iodide. While in the flow cytometry, antibodies to progesterone receptor (PR), estrogen receptor (ER), PCNA, VEGF, IL-10 and TGF-ß1 were used. For steroidogenic pathway hormones, an ELISA assay was performed. RESULTS: The results showed that cells treated with 10 µg/mL DOXO showed a 71.64% reduction in cellular growth after 72 h of treatment. Reductions in the expression of VEGF and PCNA-3 were observed by flow cytometry in all treatments when compared to the control. The intracellular levels of ERs were also significantly increased in Group III (4.67% vs. 27.1%). Regarding to the levels of steroid hormones, significant increases in the levels of estradiol (E2) and estrone sulphate (S04E1) were observed in Groups I and III. On the other hand, Group II did not show differences in steroid hormone levels in relation to the control. We conclude that the association of DOXO with AMCs (Group III) promoted a reduction in cell growth and in the expression of proteins related to proliferation and angiogenesis in IPC-366 triple-negative cells. CONCLUSIONS: This treatment promoted ER positive expression, suggesting that the accumulated oestrogen conducted these cells to a synergistic state, rendering these tumour cells responsive to ERs and susceptible to new hormonal cancer therapies.

Effects of soya milk on reproductive hormones during puberty in male Wistar rats.

Puberty is considered a critical period on development that involved sexual maturation and morphological changes. Isoflavones have been described as endocrine disruptors in male rats. Therefore, the present study attempt to evaluate the effect that daily intake of low and high doses of isoflavones exert into the hormonal regulation that take place during puberty by analyzing the levels of serum and testes steroid and pituitary hormones. 108 male pre-puberal Wistar rats (30 days old) were randomly divided into three groups; control, low and high doses of isoflavones. Experimental animals were daily dosed orally with low and high doses of a mixture of two soy isoflavones (genistein and daidzein) during 6 weeks. An EIA was performed in serum and testes homogenates for analyzing FSH, LH, P5, P4, DHEA, A4, T, DHT, SO4E1 and E2 hormone concentrations. Results revealed a decrease of an oestrogen environment in testes stimulates the secretion of FSH and LH leading to the production of androgens in the testes at the onset of puberty. Low doses of isoflavones resulted in a significant increase of testes oestrogens that consequently produced a delay on the onset of puberty; however at high doses of isoflavones the maintained oestrogenic environment in the testes prevent the stimulation of the secretion of pituitary hormones and the production of T abolishing the onset of puberty. These results clarify the hormonal mechanisms that take place on puberty and determine the effect of high and low doses of isoflavones at the onset of puberty.

Support vector machines for explaining physiological stress response in Wood mice (Apodemus sylvaticus).

Physiological stress response is a crucial adaptive mechanism for prey species survival. This paper aims to identify the main environmental and/or individual factors better explaining the stress response in Wood mice, Apodemus sylvaticus. We analyzed alterations in fecal glucocorticoid metabolite (FCM) concentration - extensively used as an accurate measure of the physiological stress response - of wild mice fecal samples seasonally collected during three years. Then, support vector machines were built to predict said concentration according to different stressors. These statistical tools appear to be particularly suitable for small datasets with substantial number of dimensions, corroborating that the stress response is an extremely complex process in which multiple factors can simultaneously partake in a context-dependent manner, i.e., the role of each potential stressor varies in time depending on other stressors. However, air-humidity, temperature and body-weight allowed us to explain the FCM fluctuation in 98% of our samples. The relevance of air-humidity and temperature altering FCM level could be linked to the presence of an abundant vegetation cover and, therefore, to food availability and predation risk perception. Body-weight might be related to the stress produced by reproduction and other intraspecific relationships such as social dominance or territorial behavior.

Steroid Tumor Environment in Male and Female Mice Model of Canine and Human Inflammatory Breast Cancer.

Canine inflammatory mammary cancer (IMC) shares clinical and histopathological characteristics with human inflammatory breast cancer (IBC) and has been proposed as a good model for studying the human disease. The aim of this study was to evaluate the capacity of female and male mice to reproduce IMC and IBC tumors and identify the hormonal tumor environment. To perform the study sixty 6-8-week-old male and female mice were inoculated subcutaneously with a suspension of 10(6)IPC-366 and SUM149 cells. Tumors and serum were collected and used for hormonal analysis. Results revealed that IPC-366 reproduced tumors in 90% of males inoculated after 2 weeks compared with 100% of females that reproduced tumor at the same time. SUM149 reproduced tumors in 40% of males instead of 80% of females that reproduced tumors after 4 weeks. Both cell lines produce distant metastasis in lungs being higher than the metastatic rates in females. EIA analysis revealed that male tumors had higher T and SO4E1 concentrations compared to female tumors. Serum steroid levels were lower than those found in tumors. In conclusion, IBC and IMC male mouse model is useful as a tool for IBC research and those circulating estrogens and intratumoral hormonal levels are crucial in the development and progression of tumors.

Circadian rhythm of salivary cortisol secretion in female zoo-kept African elephants (Loxodonta africana).

Salivary samples were collected over a 24-hr period from one group of six juvenile (7-12 years) and one group of three adult (24-25 years) African elephant females, Loxodonta africana, and the cortisol concentration was measured in unextracted samples by EIA. Samples were collected during May, June, and November 2012 (n = 147) using cotton swabs at 4-hr intervals from 20:00 to 20:00 of the next day (seven samples per animal in each trial). The animals are kept under standard zoo management: the herd is maintained in their indoor enclosures until 10:00 and then released into the outdoor enclosures until 21:00-21:30 (May/June) and 18:30-19:00 (November). No adult elephant bull was present at the zoo during this time. The results demonstrate a clear diurnal pattern of cortisol secretion with the lowest concentration observed at 20:00 (2.03 ± 0.08 ng/ml saliva) and the peak concentrations at 08:00 (5.26 ± 0.35 ng/ml saliva). Although the cortisol values were higher in the adult cows compared to the juvenile cows in the May-June period, the differences were not significant. However, the values obtained in November from the juvenile group were significantly higher (P < 0.05) than the concentrations measured in this group in June. In conclusion, salivary cortisol in zoo elephants follows a circadian rhythm (sleep-wake cycle) adapted to daily zoo husbandry routines.

Steroid hormone secretion in inflammatory breast cancer cell lines.

Inflammatory breast carcinoma (IBC) is a special type of breast cancer with a poor survival rate. Though several IBC cell lines have been established, recently a first IMC cell line was established. The aims of this study were: (1) to validate a highly sensitive, reliable, accurate and direct amplified enzyme immunoassay (EIA) to measure several cell-secreted steroid hormones: progesterone (P4), androstenedione (A4), testosterone (T), 17ß-estradiol (E2) and estrone sulfate (SO4E1) in the culture medium. (2) To assess whether hormone production profile by IPC-366 cells validates the IMC model for human IBC. We validated a non-competitive amplified EIA for inflammatory breast cancer cell lines based on the results of accuracy, precision, sensitivity and parallelism. The low detection limits of the technique were: P4=13.2 pg/well, A4=2.3 pg/well, T=11.4 pg/well, E2=1.9 pg/well and SO4E1=4.5 pg/well. Intra- and inter-assay coefficient of variation percentages were <10%. The mean recovery rate of hormone added to the culture medium was >90%. In all hormones studied SUM149 have higher levels (1.4 times, but not significant) than IPC-366, and the correlation index between SUM149 and IPC-366 concentrations were >97%. We can coclude that cells of both cell lines, IPC-366 and SUM149, are capable to produce steroid hormone in culture media. The presented EIA methodology is very valuable for the detection of steroid production in culture media and could be used in hormone regulation studies and therapeutic agents in cell lines of inflammatory and non-inflammatory mammary carcinoma or other cancer cell lines in preclinical studies.