Endocrine disruption has become a critical ecological problem, with environmental pollutants such as pesticides, industrial chemicals, and pharmaceutical residues playing crucial roles in altering endocrine regulation and disrupting key physiological processes. This overview addresses the mechanisms of endocrine disruption, focusing on how endocrine-disrupting chemicals (EDCs) interfere with hormone synthesis, receptor binding, and cellular signalling pathways, ultimately impacting animal reproductive function, development, and metabolism and thus affecting individuals, populations and ecosystems. We summarize the current state of knowledge on endocrine disruption and propose new research topics to clarify the effects of various substances with endocrine activity on organisms. Inter-species differences in sensitivity to EDCs, differences in receptor structure between species, observational studies on developmental disorders, effects of mixtures of EDCs, studies on epigenetic and transgenerational effects, and interactions between EDCs and other environmental stressors are presented as important topics for further research. Finally, we emphasize the need for collective action to protect biodiversity by curbing environmental pollution with EDCs.

Aminoglycoside antibiotics (AGs) are widely used in human and veterinary medicine. After administration, AGs undergo minimal metabolism and are excreted in their active form, contributing to their presence in the environment. The aim of our study was to determine the ecotoxicological characteristics of five AGs commonly used in human and veterinary medicine: gentamicin, neomycin, spectinomycin, streptomycin and dihydrostreptomycin. Their effects on growth and chlorophyll synthesis were investigated under laboratory conditions using the model plant organism Lemna minor. For each AG, logarithmic dilution was performed to yield test solutions with concentrations ranging from 0.001 to 100 mg/L. In the growth inhibition test, low concentrations of spectinomycin and streptomycin stimulated the growth of Lemna minor. The lowest EC50 values, indicating the greatest toxicity, were detected for gentamicin and dihydrostreptomycin, at 0.397 and 0.625 mg/L, respectively. The results of the chlorophyll synthesis test revealed that neomycin and gentamicin had the lowest EC50 values, at 45.33 and 56.01 mg/L, respectively. The other AGs tested did not notably affect chlorophyll synthesis.

BackgroundMountain gazelle (Gazella gazella) is a medium-sized antelope native to arid regions that is currently listed as an endangered species. There are only two known populations of mountain gazelles worldwide, one of which is in Hatay (Türkiye). In this study, we investigated the differences in the fecal concentrations of testosterone and progesterone metabolites in free-ranging and captive mountain gazelle.MethodsFecal samples were collected from the ground in the Hatay Mountain Gazelle Wildlife Development Area, located in Hatay Province (Türkiye) during each season of the year. In total, 246 samples, 170 from free-ranging population and 76 from captive population, were collected and used to determine testosterone and progesterone metabolite concentrations. The metabolites were extracted from dried fecal samples using methanol, and their concentrations were quantified using ELISA. The detection methods were partially validated. The analytical validation includes the determination of coefficients of variation, sensitivity of the measurements, recovery rate, linearity and cross-reactivity. In biological evaluation, the predicted reproductive status of the animals was compared with the concentrations of the progesterone and testosterone metabolites in feces.Results and conclusionOur analysis revealed that fecal samples obtained from free-ranging individuals consistently contained higher levels of testosterone metabolites than those obtained from the captive individuals. No consistent pattern was detected for fecal progesterone metabolites. Our results suggest that a constant supply of water stimulates intestinal transit. Therefore, due to faster intestinal transit the population with continuous water availability throughout the year (captive population) has lower concentrations of fecal hormone metabolites. These findings are relevant not only for the mountain gazelle as a species of endangered status but also provide important information regarding the mechanisms underlying the dynamics of steroid hormone excretion in ruminants.

IntroductionThe aim of our study was to measure fecal glucocorticoid metabolite (FGM) concentrations in captive and free-ranging male and female mountain gazelles (Gazella gazella) during their circannual cycle. In addition, FGM concentrations were used to track the intensity of the adrenocortical response in mountain gazelles during the same period.MethodsFecal samples were collected from the ground in the Hatay Mountain Gazelle Wildlife Development Area in the Hatay Province of Türkiye (36°32’ N, 36°32′ E) in each season of the year (December, April, July, September). The sex of the animals was determined by detecting the SRY gene of the Y chromosome in DNA isolated from the fecal samples. FGM was extracted from dried fecal samples with methanol, and its concentration was measured using a previously partially validated ELISA.Results and discussionThe results indicate that season is the most important factor explaining the variability in FGM concentrations in mountain gazelles. In animals of both sexes, the highest concentrations of FGM were observed in September. The values were significantly higher in the captive population, perhaps due to unpredictable stress. In July, FGM concentrations were low in both populations. As a result of the overall analysis across seasons, the comparison of FGM concentrations between captive and free-ranging animals revealed higher concentrations in captive animals only in September but not in other seasons, although higher concentrations have been previously reported for several wild captive species. Due to predation risk, the presence of offspring can be considered a critical point in the biological cycle for the welfare of free-ranging mountain gazelles, as suggested by the higher FGM concentrations in the free-ranging population in July. The high number of visitors could be a challenge for mountain gazelles in captivity, as indicated by higher FGM concentrations during September. Sex had no effect on the FGM concentrations of either population.

Dementia, especially Alzheimer's disease, presents a major clinical challenge, and researchers are still searching for an optimal animal model. To address this gap, we compared male and ovariectomized female C57BL/6 mice treated with 30 mg/kg aftin-4, which induces neurodegeneration, with naturally aged (15-16 months old) mice not treated with aftin-4. We performed a series of behavioral tests; measured postmortem plasma beta-amyloid levels (A beta 1-40 and A beta 1-42) and the levels of the oxidative stress indicators glutathione peroxidase (GPx), superoxide dismutase (SOD) and malondialdehyde (MDA); and evaluated astrocytic reactivity in the brain using glial fibrillary acid protein (GFAP) levels. Our results revealed no behavioral changes in the aged or aftin-4-treated mice compared with the control mice. Aftin-4 mice presented lower brain MDA levels and no detectable changes in plasma A beta levels. In general, female mice had higher GPx and SOD levels and lower A beta 1-42 levels than male mice did. In contrast, aged and aftin-4-treated male mice presented elevated levels of GFAP, indicating astrocyte damage. Our results could not confirm that either aftin-4-treated or aged mice are reliable models for dementia. However, the observed molecular changes suggest that male animals may be more susceptible to oxidative stress and brain damage than females are. This study demonstrates the complexity of modeling dementia in animals and the importance of future studies in this area.

IntroductionMeasurement of hair cortisol concentration (HCC) is a useful tool for assessing the activity of the hypothalamic-pituitary-adrenal axis and thus evaluating the long-term adrenocortical response in different animal species and breeds. Robust indigenous pig breeds are highly adapted to the local environment and are preferred for organic farming, compared to the commercial breeds. We investigated whether seasonality, breeding system (indoor or outdoor) and sex influence HCC of pigs reared on organic farms.Materials and methodsFifty-three pigs of the indigenous Slovenian Krškopolje breed were divided into three groups. Group Ind (n = 15) was housed indoors, groups Out-1 (n = 18) and Out-2 (n = 20) were housed outdoors on two different farms for the duration of 9 months. Hair was sampled once per season in the withers area of each pig and HCC was determined using a previously validated ELISA.Results and discussionThe effect of seasonality was found to be significant and more pronounced in pigs reared outdoors. HCC were highest and varied most in winter for all groups, while they were lowest and varied less in summer and autumn. The highest HCC was measured in group Out-1 in winter, as it was the only group housed outdoors at that time. Group Ind had significantly higher HCC in summer compared to the groups Out-1 and Out-2, which could be due to hair cortisol degradation by the UV light exposure in outdoor groups. Sex had no effect on HCC. Our study suggests that seasonality and housing type influence the HCC of pigs.

Increasing use of pigs as models in translational research, and growing focus on animal welfare are leading to better use of effective analgesics and anaesthetics when painful procedures are performed. However, there is a gap in basic knowledge such as pharmacokinetics of different anaesthetics in these species. The main objective of our study was to determine the pharmacokinetics of levobupivacaine in domestic pigs. Twelve female grower pigs weighing 31.17 ± 4.6 kg were subjected to general anaesthesia and experimental surgery, at the end of which they received 1 mg/kg levobupivacaine via peri-incisional subcutaneous infiltration. Plasma samples were collected before administration of levobupivacaine and at 0.5, 1, 2, 4, 8, 12, 24 and 48 h thereafter. Concentrations of levobupivacaine were determined by liquid chromatography coupled with tandem mass spectrometry. Following single dose of levobupivacaine, all animals had measurable plasma concentrations 0.5 h after drug administration, with most peak concentrations observed at the 1-h time point. In all 12 animals, levobupivacaine was below the limit of quantification 48 h after drug administration. The mean maximum plasma concentration, area under the curve and half-life were determined to be 809.98 μg/l, 6552.46 μg/l h and 6.25 h, respectively. Plasma clearance, volume of distribution and weight-normalized volume of distribution were 4.41 l/h, 35.57 l and 1.23 l/kg, respectively. Peak plasma concentrations in our study were well below concentrations that were found to produce toxicity in pigs.

Animal welfare is important; therefore, veterinarians and other animal welfare experts try to use different tools for pig welfare assessment. Several welfare protocols are available for pig welfare assessment, and one of the most used is Welfare Quality (WQ) protocol®. Elevated values of cortisol can be indicative of stress and, therefore, poor welfare. Our aim was to assess the correlation between serum cortisol levels from individual samples and oral fluid cortisol levels in group samples with the grades received for pig welfare using the WQ protocol®. Samples were taken at six different commercial pig farms. Animals were divided into age-dependent categories: 5 weeks old (w/o); 7 w/o; 9 w/o; 11 w/o weaners; fatteners; and breeding sows (10 pigs/category). Cortisol was determined in individual sera and group samples of oral fluid (OF), and was compared to values considered to be physiological. Based on WQ protocol® answers, five farms’ welfare level was deemed acceptable, and one was enhanced. Four out of 29 sera and 5 out of 30 OF samples were considered physiological, while in most other samples it was elevated. The correlation between cortisol levels in sera, OF, and WQ protocol® scores was not statistically significant. The cortisol level in OF should be just one of the welfare indicators, i.e., alongside the WQ protocol® filled out by a welfare expert.

The stress response, which involves joint activity of the nervous and endocrine systems, is one of the basic adaptive mechanisms that ensures the survival of the individual. The activation of the sympathetic nervous system, the sympathetic-adrenal-medullary axis, and the hypothalamic–pituitary–adrenal axis enables organisms to respond to endogenous and exogenous challenges. Repeated short-term stress leads to long-term stress, which disrupts physiological homeostasis. Unlike domestic animals, wild animals are not protected from environmental and weather influences or treated for diseases. In addition, climate change, habitat fragmentation and loss, and urban stressors (such as light, noise and chemical pollution; xenobiotics; traffic; and buildings) affect individual wildlife and populations. In this review, we have attempted to depict the magnitude of the stress response in wildlife and related domestic animals as well as in captive and free-ranging animals. The intensity of the stress response can be estimated by determining the concentration of glucocorticoids in body fluids, tissues, and excreta. A comparison of results from different studies suggests that domestic animals have lower fecal and hair glucocorticoid concentrations than related wild animals. Additionally, fecal and hair glucocorticoid concentrations in captive animals are higher than in free-ranging animals of the same species. As there are limited data on this topic, we cannot draw definitive conclusions about glucocorticoid concentration and stress response. Further studies are needed to clarify these issues.

The cholinergic system is involved in the regulation of all organ systems and has acetylcholine (ACh) as almost its only neurotransmitter. Any substance is called cholinergic if it can alter the action of acetylcholine. Cholinesterases (ChEs) are enzymes that enable the hydrolysis of acetylcholine and in this way ensure homeostasis in cholinergic synapses. Cholinesterase inhibitors (ChEi) are a group of indirect-acting cholinergic agonists that influence the activity of the cholinergic system. Several compounds that can inhibit cholinesterases are of importance to veterinary medicine from pharmacological and toxicological perspective. The frequency of their use in veterinary medicine has fluctuated over the years and is now reduced to a minimum. They are mainly used in agriculture as pesticides, and some are rarely used as parasiticides for companion animals and livestock. In recent years, interest in the use of new cholinesterase inhibitors has increased since canine cognitive dysfunction (CCD) became a recognized and extensively studied disease. Similar to Alzheimer's disease (AD) in humans, CCD can be treated with cholinesterase inhibitors that cross the blood–brain barrier. In this review, the mammalian cholinergic system and the drugs that interact with cholinesterases are introduced. Cholinesterase inhibitors that can be used for the treatment of CCD are described in detail.

Comprehensive comparisons of similar lipid based drug delivery systems produced by different technologies are scarce. Spray drying and fluid bed layering technologies were compared with respect to the process and product characteristics of otherwise similar simvastatin loaded dry emulsion systems. Fluid bed layering provided higher process yield (83.3% vs 71.5%), encapsulation efficiency (80.0% vs 68.4%), relative one month product stability (93.8% vs 85.5%), larger and more circular particles (336 µm vs 56 µm) and lower median oil droplet size after product reconstitution in water (2.85 µm vs 4.27 µm), compared to spray drying. However, spray dried products exhibited higher drug content (22.2 mg/g vs 9.34 mg/g). An in-vivo pharmacokinetic study in rats was performed and a pharmacokinetic model was developed in order to compare the optimised simvastatin loaded dry emulsion systems, a simvastatin glyceride mimetic loaded in the dry emulsion and a simvastatin loaded SMEDDS with a reference physical mixture. Of the formulation tested, fluid bed layered pellets excelled and provided a 115% relative increase in bioavailability. Among the two technologies, fluid bed layering provided dry emulsion products with higher relative bioavailability and better product characteristics for further processing into final dosage forms.

Thyme (Thymus vulgaris L.) is a well-known medicinal plant, the aerial parts of which have long been used internally or externally as a traditional remedy for various diseases. Thyme essential oils have important pharmaceutical applications and are regularly used in the pharmaceutical, food and cosmetic industries. In folk medicine, thyme preparations are used to treat respiratory, digestive, cardiovascular and nervous disorders, as well as to relieve dysmenorrhea. Thymol, a major constituent of Thymus vulgaris essential oil, has been shown to affect skeletal and smooth muscle excitation and contraction. Therefore, the main objective of this study was to evaluate its effect on acetylcholine (ACh)-induced rat intestinal and uterine preparations. Isolated ileum and uterine horn preparations were placed in a 20-ml organ bath containing Tyrode or De Jalon solution and exposed to graded concentrations of ACh (0.036, 0.109, 0.36, 1.09, 3,6, 10.9, and 36 μM) and either 0.02 or 0.2 mM thymol. Additionally, the dose–response relationship of thymol impact on intestinal and uterine contraction was evaluated. Contraction changes were monitored using an isometric transducer. Thymol at the higher dose (0.2 mM) significantly reduced ACh-induced intestinal and uterine contractions. Thus, this study provides new important data on competitive actions between thymol and ACh. In the dose–response study, the IC50 values were calculated as 5.26 mM for the ileum and 5.35 mM for the uterus. Our results demonstrated the efficacy of thymol in reducing ileal and uterine smooth muscle contractions, thus supporting the use of thyme in traditional medicine in the treatment of digestive disorders and painful menstrual cramps.

Background: Beside others, neuroinhibitory and sedative effects of CBD were documented. Aim and Methods: The aim of the study was to assess the dose-related effects of CBD premedication on the course of isoflurane anesthesia. Wistar rats were pretreated with different doses of CBD 1 h before isoflurane anesthesia. In the pretreatment, animals were given CBD at doses of 100, 20, 10, or 2 mg kg-1. Before the fifth (control) anesthesia, the animals were given only mid-chain triglyceride oil, which served as a solvent in the CBD formulation. The induction time was determined, and on awakening, the time to appearance of the flexion reflex and the recovery from anesthesia were determined. Results: Statistical analysis showed a significantly shorter induction time if animals were pretreated with 20 mg kg-1 CBD. In addition, pretreatment with 100 mg kg-1 CBD resulted in a prolonged induction time, while on awakening, delayed appearance of reflexes and prolonged recovery from anesthesia compared to pretreatment with 20 mg kg-1 CBD were observed. Conclusions: The results indicate that the influence of CBD on the course of isoflurane anesthesia depends on the dose and can reduce the induction time. Although this study was performed in laboratory rats, in clinical practice, these data should be considered when CBD-treated patients undergo isoflurane anesthesia.

Abstract Bee products produced in environments with persistent organic pollutants (POPs) may be contaminated by these compounds. Propolis usually consists of beeswax, resins, water, inorganic and phenolic substances, and essential oils. Although there are some studies on the detection of POPs in honey and some bee products, these are quite limited. Because propolis is a botanical-based and highly complex matrix, and thus difficult to analyze, a reliable and selective method based on solid-phase extraction (SPE) combined with gas chromatography-mass spectrometry was developed for the determination of organochlorine pesticides (OCPs), polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) in the propolis. All analytes were extracted with acetonitrile and centrifuged for 10 min at 4000 rpm and 6 °C in a refrigerated centrifuge. The samples were filtered using a homemade SPE cartridge containing C18, primary-secondary amine, and magnesium sulfate. The residue collected with acetonitrile was subsequently analyzed by gas chromatography – mass spectrometry (GC-MS). The proposed method was validated according to the Eurachem guidelines and applied directly to the collected propolis samples. The results show that the method may be used successfully in residue monitoring laboratories for the determination of OCPs, PCBs and PBDEs in propolis.

Welfare of dairy cows can be assessed using welfare assessment protocols consisting of resource, management and animal-based measures. Welfare Quality® Assessment Protocol is one of the best-known protocols, which depends almost entirely on animal-based measures. To gain more objective and rapid welfare assessment, new techniques have been developed to measure welfare of animals, such as hair cortisol concentration. As cortisol is released in response to stress, it has long been used as a biomarker of stress in animals. While the precise mechanism of cortisol incorporation into hair is unknown, hair cortisol concentration seems to be a marker of long-term systemic cortisol concentration. Hair cortisol is, therefore, a potential marker of chronic stress and is not likely to be affected by acute stress. Studies on cattle show connections between hair cortisol concentration and factors such as pregnancy, parity, diseases, ectoparasites, body condition score, environmental changes, stocking density and milk yield. Hair cortisol concentration appears to be affected by time of sampling, cow age and breed, UV radiation, season, body region of sampled hair and hair colour. Its concentration also depends on sampling and analytical methods. Hair cortisol is a promising non-invasive tool to evaluate welfare of dairy cows, however, more research is needed to determine the extent of effects on its concentration and the appropriate method of sampling and analysis. Correlations between Welfare Quality® Assessment Protocol scores and pooled hair cortisol concentrations have not yet been found, and more research is needed with larger sample size, a standardized protocol of hair sampling, processing and analysis. With proper attention to detail, hair cortisol levels in pooled hair samples might come to be used as a reliable indicator of dairy animal welfare.

Background Early maternal separation may have long-lasting physiological effects on different organ systems. Although long-lasting effects of early maternal separation are mostly studied in connection with the development and function of the central nervous system hypothalamic–pituitary–adrenal axis, animal health, behaviour and productivity, there is a lack of information about its impacts on the reproductive system. In the dairy industry, calves are often separated from mothers 1 or 2 days after birth and are not nursed. In the present retrospective study based on data from an artificial insemination (AI) centre, we compared semen parameters and fertility in bulls that were separated from their mothers one day after birth with the semen parameters of bulls that remained with their mothers and were nursed for approximately 2 months. Semen parameters were followed in 3 consecutive years in 52 maternally separated and 22 nursed bulls. Results Ejaculate volume and total sperm count in ejaculate were significantly higher in nursed bulls in comparison to maternally separated bulls at the age 25–36 and 37–48 months, but interestingly, not at the age 12–24 months, during the first year in the AI centre. Non-return rates did not differ between separated and nursed bulls. Conclusion The results suggest that early maternal separation causes long-lasting effects on the functioning of the male reproductive system, evident by reduced production of semen in adult bulls. The data suggest that with a standard of 20 million sperms per straw of frozen semen, 27–78 fewer straws can be obtained from one ejaculate of maternally separated bulls in comparison to the nursed bulls.

In the last 60 years, milk, meat, and egg worldwide consumption per capita increased 2-, 3-, and 5-fold, respectively. In the same period, several studies have reported a drop of semen quality and an increased incidence of cryptorchidism, hypospadias, and some hormone-related cancers (testicular, prostate, breast, endometrial cancer) in humans. A possible reason for these disruptions is increased exposure to exogenous hormones in food of animal origin. Hormones in food are considered to affect the endocrine system and cell signaling and thus disrupt homeostasis in the consumers. Since food safety assurance is a part of the veterinary service, the risk assessment of hormones in food as potential disruptive compounds is a significant challenge. Numerous cohort and epidemiologic studies and meta-analyses have been performed with respect to find an association between the consumption of food of animal origin and particular health disorders. Additionally, several studies in animal models have been performed to explain the mechanisms of disruptive effects of exogenous hormones. Since contradictory results have been reported, there is no general conclusion about the disruptive effects of exogenous hormones. Further experiments on animals in which long-term treatment with exogenous hormones is performed and further comprehensive endocrinological, toxicological, and human epidemiological studies are needed to confirm or deny the role of exogenous hormones in human health disorders. Moreover, long-term exposure to a combination of several exogenous compounds, such as environmental pollutants and dietary hormones and their additive effects, are also not well known and should be a topic for further studies.

Extracts of sweet chestnut (Castanea sativa Mill.) which contain hydrolysable tannins are used to prevent diarrhoea in farm animals due to the astringent effect of tannins. The aim of this study was to establish whether long-term treatment with the chestnut extract as a feed additive affects the acetylcholine (ACh)-triggered contractions of isolated rat ileum. Wistar rats of both sexes were divided into 4 male and 4 female groups with 6 animals in each group. The first, second and third groups of both sexes received feed with 3, 15 and 30 g of chestnut extract per kg, respectively. The fourth group of both sexes received feed without the added chestnut extract and served as the control group. After the 90-day trial, the animals were euthanized, and ilea were isolated and placed in an organ bath filled with Tyrode's solution. Ilea were connected to an isometric transducer. Ileal contractions were triggered by 0.005, 0.01, 0.05, 0.1, 0.5, 1.0, 5.0 and 10.0 mg of ACh, and contractions were recorded. The groups exhibited dose responsiveness to the ACh treatment steps; however, no significant differences in the contraction intensity were observed among the groups at the same ACh dose level. These results suggest that long-term treatment with chestnut extract as an astringent does not influence the intensity of ileal contractions triggered by ACh. Thus, no adverse effect on intestinal motility can be expected during a long-term use of chestnut extract as a feed additive in livestock production; however, further studies in target animal species are needed to confirm the findings.

The aim of this study was to compare hair corticosteroid concentrations in ectoparasiticide-treated (n = 10) and non-treated (n = 12) Holstein cows. Animals in the treated group received cyfluthrin three times, on days 0, 28 and 56 of the experiment. Hair samples were collected from all cows on days 0, 21, 42, 63, and 84 of the experiment for the determination of cortisol and corticosterone concentrations using immunoassays. The respective hair cortisol concentrations in the treated group averaged 209.78, 165.10, 260.78, 177.44 and 183.11 ng/g, while in the non-treated group hair cortisol concentrations averaged 172.58, 243.58, 309.73, 199.75 and 207.09 ng/g. These results indicate that the control of ectoparasites reduced hair cortisol levels in dairy cows. The respective hair corticosterone concentrations in the treated group averaged 19.06, 22.95, 21.95, 21.60 and 24.84 ng/g and in the non-treated group the hair corticosterone concentrations averaged 17.28, 21.94, 34.05, 26.27 and 26.91 ng/g. The results suggest that longterm stress can be estimated better by the determination of hair cortisol rather than corticosterone concentrations.