Welfare assessment in dairy cows using hair cortisol as a part of monitoring protocols.

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.

Injections to pregnant mice produce prenatal stress that affects aggressive behavior in their adult male offspring.

Maternal stress could reprogram the developing fetal nervous system. A common target of maternal glucocorticoids is fetal neuro-endocrine axis. In the present study, pregnant mice were exposed to stress by injection and their male offspring were tested for sexual and aggressive behaviors in adult life. Three groups of pregnant mice were exposed to stress by sham syringe injection. The first group was injected on days 13, 14, and 15 p.c., the second group was injected on days 17 and 18 p.c., and the third group was injected daily from days 13 to 18 p.c. while control mice were not injected. Male offspring that were exposed to stress on days 13-18 p.c. and 17-18 p.c. were less aggressive and had lower blood testosterone levels in comparison to the control group. In male sexual behavior, there were no statistically significant differences between the groups. Body weight differed significantly with groups injected on days 13-18 p.c. and 13-15 p.c. having significantly higher body weight in adult life than the other two groups. After behavioral testing, brains were processed for immunohistochemical staining with antibodies against vasopressin (AVP) and calbindin (CALB). The expression of AVP and CALB in the lateral septum and in the preoptic area, respectively, did not differ between groups, suggesting that these two masculinization markers were not affected by prenatal stress. Present study therefore shows that even presumably mild and short prenatal stress weakens aggressive behavior of adult male mice, possibly due to reduced testosterone levels in blood.

Preventing ectoparasite infestations reduces glucocorticoid concentrations in the hair of cows - short communication.

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.

Gonadectomy prior to puberty decreases normal parental behavior in adult mice.

Sex steroid hormones secreted by gonads influence development and expression of many behaviors including parental behaviors. The capacity to display many behaviors develops under the influence of sex steroid hormones; it begins with gonadal differentiation and lasts through puberty. The timing of gonadectomy may have important and long lasting effects on the organization and activation of neural circuits regulating the expression of different behaviors. The present study investigated the importance of exposure to endogenous gonadal steroid hormones during pubertal period/adolescence on parental behavior in adult mice. Male and female WT mice were gonadectomized either before puberty (25 days of age) or after puberty (60 days of age) and tested for parental behavior with and without estradiol benzoate (EB) replacement in adulthood. Additional groups of mice were gonadectomized at P25 and supplemented with estradiol (females) or testosterone (males) during puberty. Female mice gonadectomized after puberty or gonadectomized before puberty and supplemented with estradiol during puberty, displayed better pup directed parental behaviors in comparison to mice gonadectomized at 25 days of age regardless of treatment with estradiol in adulthood. However, mice treated with EB in adulthood displayed better non-pup directed nest building behavior than when they were tested without EB treatment regardless of sex and time of gonadectomy. To examine whether the sensitivity to sex steroid hormones was altered due to differences in time without gonads prior to the testing, mice were also tested for female sex behavior and there were no differences between mice gonadectomized at P25 or P60, although this could not completely rule out the possibility that parental behavior is more sensitive to prolonged absence of steroid hormones than female sex behavior. These results suggest that the absence of gonads and thereby the absence of appropriate gonadal steroid hormones during puberty/adolescence may have a profound effect on pup directed parental behaviors in adult mice.

Fluctuations of serum cortisol, insulin and non-esterified fatty acid concentrations in growing ewes over the year.

BACKGROUND: The physiological levels of endocrine and metabolic parameters in Slovene autochthonous breeds of sheep are not yet well known, nor are the mechanisms of their adaptability and responses to climate and environmental factors. Therefore, the aim of this study was to evaluate fluctuations of cortisol, insulin and non-esterified fatty acids (NEFA) in growing ewes over an one-year period. Blood samples were collected monthly from 10 yearling Jezersko-Solchava, 10 Bovec and 10 Istrian ewes. Serum cortisol, insulin and NEFA were measured with commercial kits. RESULTS: Mean monthly cortisol values fluctuated with low levels in summer and high levels in autumn. Significant peaked cortisol values of 25.69 ± 6.89, 14.67 ± 2.43 and 21.11 ± 7.25 µg/L in Jezersko-Solchava, Bovec and Istrian breed, respectively, were found in September (Bovec breed) and October (Jezersko-Solchava and Istrian breed). Mean monthly insulin values increased during the observation period. The highest levels of 14.60 ± 3.15, 16.03 ± 5.35 and 12.56 ± 2.52 µIU/mL in Jezersko-Solchava, Bovec and Istrian breed, respectively, were observed in the last sample collection in May. NEFA concentrations were found to be low except in some autumn and spring months. The peak values were observed in March for Jezersko-Solchava and Istrian breed (0.60 ± 0.05 and 0.66 ± 0.10 mmol/L), and in April for Bovec breed (0.71 ± 0.11 mmol/L). CONCLUSIONS: Monthly fluctuations of cortisol, insulin and NEFA were measured in all observed sheep breeds, but between-breed differences in monthly values of examined parameters were insignificant. Significantly increased serum cortisol levels were found in autumn for all breeds and were probably associated with the onset of puberty and low environmental temperature. A gradual increase of insulin level in the examined ewes was in parallel with their growth. Significantly higher NEFA values in spring suggest qualitatively insufficient feed supply during that period.

Effects of season and sex on the concentrations of fecal glucocorticoid metabolites in captive and free-ranging endangered mountain gazelles (Gazella gazella).

Introduction: The 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. Methods: Fecal 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 discussion: The 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.

Retrospective study of bull semen quality - possible correlation with pesticide use?

Decline in semen quality in humans and increased incidence of male reproductive problems could be caused by different factors, including pesticides that could mimic or block the action of endogenous hormones. If the decline in semen quality is real, and environmental chemicals are at least partially responsible for this decline, similar changes should be observed in animals that live in close connection with humans and are exposed to similar levels of pollutants. In the present study, the semen quality of bulls in the last 30 years was examined with respect to the year of birth. Furthermore, semen quality results were correlated to the total pesticide use in a limited geographical area. The results indicate a notable decrease in both ejaculate volume and total number of spermatozoa in ejaculates of bulls born in the late 1970s, while after that (until 2006) there was no obvious downward or upward trend either in ejaculate volume or in the total number of sperm cells. The amount of pesticides released into the environment increased about twofold in the given period, and linear regression analysis revealed a strong and statistically significant correlation between the amount of pesticides used and the total number of spermatozoa in the ejaculate.

Potential revival of cholinesterase inhibitors as drugs in veterinary medicine.

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.

Stress in wildlife: comparison of the stress response among domestic, captive, and free-ranging animals.

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 Impact of Cannabidiol on the Induction of Isoflurane Anesthesia and Recovery in Wistar Rats.

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.

The effect of thymol on acetylcholine-induced contractions of the rat ileum and uterus under ex vivo conditions.

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.

Relative bioavailability enhancement of simvastatin via dry emulsion systems: Comparison of spray drying and fluid bed layering technology.

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.

Calves' management conditions affect sperm count in adult bulls.

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.

MECHANISMS IN ENDOCRINOLOGY: Estrogens in consumer milk: is there a risk to human reproductive health?

Possible effects of xenoestrogens on human health, in particular on male reproductive health, have attracted considerable attention in recent years. Cow's milk was suggested in numerous publications as one of possible sources of xenoestrogens that could affect human health. Although milk has undoubtedly many beneficial health effects and could even have a role in reducing incidence of some cancers, concerns were raised about presumably high levels of estrogens in cow's milk. In intensive farming, concentrations of estrogens in milk are higher due to long milking periods that today extend long into the pregnancy, when concentrations of estrogens in the cow's body rise. Numerous studies examined potential effects of milk on reproductive health and endocrine-related cancers in both experimental studies with laboratory animals, and in human epidemiological studies. In the present review article, we compiled a review of recently published literature about the content of estrogens in cow's milk and potential health effects, in particular on reproductive system, in humans. Although results of published studies are not unequivocal, it seems that there is stronger evidence suggesting that amounts of estrogens in cow's milk are too low to cause health effects in humans.

Sex steroid levels in urine of cattle of different ages: evaluation of abuse control procedures.

Levels of several natural urinary steroids have been determined in the urine of a large number of animals of different cattle categories in the context of steroid abuse in beef production. Bovine animals of different breeds, sex and age included in the Slovene national residue detection plan for steroid abuse were studied. Urine from 120 males and 174 females was analysed. Urinary boldenone, boldione, androstenedione, equiline, medroxyprogesterone, medroxyprogesterone acetate, melengestrol acetate, progesterone, stanozolol, trenbolone, trenbolone acetate, 17α-ethinylestradiol, 17α-methyltestosterone, epitestosterone, 17ß-estradiol, testosterone, and nandrolone were determined by LC-MS/MS. Epitestosterone was found in all bulls; while the proportion of animals containing testosterone and androstenedione increased with age. Testosterone was not detected in bulls less than 5 months of age. Epitestosterone levels, however, were not age dependent. The ratio of testosterone to epitestosterone thus increased with age, from 0.13 ± 0.09 at 1-7 months to 0.42 ± 0.10 at 25-38 months. It was significantly (p < 0.01) higher in bulls above 13 months than in younger animals. In contrast to males, no urinary testosterone was found in females, whereas epitestosterone, androstenedione, progesterone and estradiol were present. The proportion of animals of various age groups in which epitestosterone was detected ranged from 68% to 100%, but the differences were not significant. The presence of both estradiol and progesterone in the same sample was not observed in any animal. The results of this study could be helpful in determining physiological urinary steroid levels in order to provide a baseline for the control of steroid abuse in beef production.