Validation of a commercial ELISA kit to measure 11-oxoetiocholanolone in equine and bovine feces.

Feces are a noninvasive and easily collectible matrix that may help determine cumulative hormone metabolite concentrations over medium-to-long times. To date, 11-oxoetiocholanolone, an important metabolite of cortisol, has been measured in equine and bovine feces solely by an in-house enzyme immunoassay (EIA). Therefore, we validated the use of a commercial ELISA kit (11-oxoetiocholanolone ELISA kit; Cayman Chemical), which had been validated on sheep feces and human urine, to measure 11-oxoetiocholanolone in feces from 42 horses and 32 bulls. The ELISA kit had good precision (intra- and inter-assay CVs: 5.8% and 11.2% for equine feces; 9.9% and 11.2% for bovine feces, respectively), analytical sensitivity (0.186 ng/mL for both equine and bovine feces), and accuracy (parallelism and recovery tests) in determining 11-oxoetiocholanolone concentrations in feces from both species. We found ranges of 11-oxoetiocholanolone concentrations of 1-109 ng/g in equine feces and 40-302 ng/g in feces of bulls. The Cayman Chemical ELISA kit offers a simple and accessible means of analysis of 11-oxoetiocholanolone in equine and bovine fecal samples.

Peripubertal Testosterone, 17ß-Estradiol and Progesterone Concentrations in Hair and Nails in Dobermann Dogs.

Studies about puberty in dogs are few, probably because many factors are involved in the delicate process of puberty onset, leading to difficulties in the proper enrollment of subjects. Moreover, the use of blood for monitoring hormonal changes can be problematic, and not feasible for long-term studies. Hair and nails proved to be suitable matrices for the retrospective evaluation of hormones' long-term accumulation. This study was performed using hair and nails for the evaluation of testosterone (T), 17ß-estradiol (E2) and progesterone (P4) concentrations to assess possible sexual steroid changes during the peripubertal period in dogs. The results, obtained on five males and five females, showed a significant increase in T in hair and nails of males immediately before puberty. In females, a significant increase in E2 at puberty and a marked increase in P4 after puberty was found in both biological specimens. Sex-related differences were detected only for T hair concentrations, but when the sex and sampling time were considered together, hair and nails T and nails P4 concentrations allowed us to discern between male and female dogs at specific sampling times. The results from this study showed that hair and nails are useful biological specimens for the retrospective evaluation of changes in T, E2 and P4 concentrations in peripubertal dogs.

Is Smart Working Beneficial for Workers' Wellbeing? A Longitudinal Investigation of Smart Working, Workload, and Hair Cortisol/Dehydroepiandrosterone Sulfate during the COVID-19 Pandemic.

Building on the job demands-resources (JD-R) and allostatic load (AL) models, in the present study we examined the role of smart working (SW) in the longitudinal association between workload/job autonomy (JA) and a possible biomarker of work-related stress (WRS) in the hair-namely, the cortisol-dehydroepiandrosterone sulfate (DHEA(S)) ratio-during the COVID-19 pandemic. Overall, 124 workers completed a self-report questionnaire (i.e., psychological data) at Time 1 (T1) and provided a strand of hair (i.e., biological data) three months later (Time 2, T2). Results from moderated multiple regression analysis showed that SW at T1 was negatively associated with the hair cortisol/DHEA(S) ratio at T2. Additionally, the interaction between workload and SW was significant, with workload at T1 being positively associated with the hair cortisol/DHEA(S) ratio at T2 among smart workers. Overall, this study indicates that SW is a double-edged sword, with both positive and negative consequences on employee wellbeing. Furthermore, our findings suggest that the hair cortisol/DHEA(S) ratio is a promising biomarker of WRS. Practical implications that organizations and practitioners can adopt to prevent WRS and promote organizational wellbeing are discussed.

Incorporation of Testicular Ultrasonography and Hair Steroid Concentrations in Bull Breeding Soundness Evaluation.

Testicular ultrasonography and steroid concentrations (cortisol, dehydroepiandrosterone sulfate (DHEA-S), cortisol/DHEA-S ratio, testosterone) in hair were examined for their utility in the bull breeding soundness evaluation (BBSE). Beef and dairy bulls (n = 16; 2.7 ± 0.4 years old; body condition score 3.2 ± 0.1) of five breeds were maintained under the same conditions at an accredited semen collection center. Bulls underwent routine semen collection twice weekly for 12 weeks and semen was processed and cryopreserved. Ultrasonography and hair sampling were undertaken at the last semen collection. Bulls with homogeneous testicular parenchyma (n = 8) had a higher (p < 0.05) percentage of motile sperm post-thawing compared with bulls with heterogeneous parenchyma (n = 8). There were no differences (p > 0.05) in the hair concentrations of cortisol, DHEA-S, and testosterone between bulls with homogeneous and heterogeneous parenchyma. In bulls with homogeneous parenchyma, hair DHEA-S concentration was positively correlated with percentage motile sperm (R2 = 0.76), progressively motile sperm (R2 = 0.70), and motility yield (R2 = 0.71). The findings indicate that the integration of testicular ultrasonography and hair DHEA-S status in the BBSE could provide a more comprehensive assessment of indicative fertility in bulls. Additionally, ultrasonography can be used in the BBSE when the evaluation of semen parameters is not available.

Validation of an ELISA kit to measure allopregnanolone in human and equine hair.

In humans, allopregnanolone plays important roles in a number of different neurodegenerative disorders, and it has been proposed for use in some therapies. Horses are commonly used as animal models for human neurodegenerative diseases, mental and behavioral disorders, and neuropsychiatric disorders, and there is interest in using hair as a biological sample to study hormones in these conditions. We validated the use of a commercial ELISA kit (DetectX allopregnanolone kit; Arbor Assays), which was designed for serum, plasma, feces, urine, and tissue samples, to assess allopregnanolone in hair samples from 30 humans and 63 horses. The ELISA kit had good precision (intra- and inter-assay CVs: 6.4% and 11.0% for equine hair; 7.3% and 11.0% for human hair, respectively), sensitivity (50.4 pg/mL for both equine and human hair), and accuracy (parallelism and recovery tests) in determining allopregnanolone concentrations in hair from both species. The allopregnanolone concentrations in human hair were 7.3-79.1 pg/mg; the concentrations were 286 ± 141 pg/mg (x̄ ± SD) in mares on the day of parturition and 16.9 ± 5.5 pg/mg in nonpregnant mares. The DetectX ELISA kit offered a simple and accessible analysis of allopregnanolone in human and equine hair samples.

Coat, Claw and Dewclaw 17-ß-Estradiol and Testosterone Concentrations in Male and Female Postpubertal Cats: Preliminary Results.

In the recent past, tissue materials such as hair/coat and nails/claws have proved to be useful for the study of long-term hormonal changes in humans and animals and shown to be advantageous in terms of being collectable without invasiveness, with a benefit in terms of animal welfare. However, studies using these tissue materials in cats are scarce, especially on sexual hormone measurement. In this study, the concentrations of 17-ß-estradiol (E2) and testosterone (T) were assessed in 20 male and 18 female domestic postpubertal cats at the time of neutering/spaying during the breeding season. Hormones were measured in coat shaved from the forearm (ACOAT) and from the surgical area (SCOAT); claws were collected from the forearms (CLAWS) and the dewclaws (DCLAWS). Although all these tissue materials were shown to be useful for E2 and T long-term measurement, only T concentrations were higher (p < 0.001) in males from both ACOAT and SCOAT samples when compared to females and therefore useful for distinguishing between the two sexes. Within each sex, E2 and T concentrations can be assessed on coat, but also on the dewclaws, providing an alternative, practical, matrix for sexual steroid measurement in postpubertal cats during the breeding season.

Hair and Claw Dehydroepiandrosterone Concentrations in Newborn Puppies Spontaneously Dead within 30 Days of Age.

The latest intrauterine fetal developmental stage and the neonatal period represent the most challenging phases for mammalian offspring. Toward the term of pregnancy, during parturition, and after birth, the hypothalamic−pituitary−adrenal axis (HPA) is a key system regulating several physiologic processes, through the production of cortisol and dehydroepiandrosterone (DHEA). This study was aimed to assess DHEA concentrations in hair and claws of 126 spontaneously dead newborn puppies, classified as premature puppies (PRE-P), stillborn puppies (STILL-P) and puppies that died from the 1st to the 30th day of life (NEON-P). The possible influence of newborn sex, breed body size, and timing of death on DHEA concentrations in both matrices was evaluated. Claw DHEA concentrations were higher in the PRE-P group when compared to STILL-P and NEON-P puppies (p < 0.05), whilst no significant differences were found in hair for all the studied factors. The results confirm the hypothesis that higher amounts of DHEA are produced during the intrauterine life in dogs, also in puppies that will die soon after birth.

Hair Cortisol and DHEA-S in Foals and Mares as a Retrospective Picture of Feto-Maternal Relationship under Physiological and Pathological Conditions.

Equine fetal hair starts to grow at around 270 days of pregnancy, and hair collected at birth reflects hormones of the last third of pregnancy. The study aimed to evaluate cortisol (CORT) and dehydroepiandrosterone-sulfate (DHEA-S) concentrations and their ratio in the trichological matrix of foals and mares in relation to their clinical parameters; the clinical condition of the neonate (study 1); the housing place at parturition (study 2). In study 1, 107 mare-foal pairs were divided into healthy (group H; n = 56) and sick (group S; n = 51) foals, whereas in study 2, group H was divided into hospital (n = 30) and breeding farm (n = 26) parturition. Steroids from hair were measured using a solid-phase microtiter radioimmunoassay. In study 1, hair CORT concentrations measured in foals did not differ between groups and did not appear to be influenced by clinical parameters. A correlation between foal and mare hair CORT concentrations (p = 0.019; r = 0.312, group H; p = 0.006; r = 0.349, group S) and between CORT and DHEA-S concentrations in foals (p = 0.018; r = 0.282, group H; p < 0.001; r = 0.44, group S) and mares (p = 0.006; r = 0.361, group H; p = 0.027; r = 0.271, group S) exists in both groups. Increased hair DHEA-S concentrations (p = 0.033) and decreased CORT/DHEA-S ratio (p < 0.001) appear to be potential biomarkers of chronic stress in the final third of pregnancy, as well as a potential sign of resilience and allostatic load in sick foals, and deserve further attention in the evaluation of prenatal hypothalamus-pituitary-adrenal (HPA) axis activity in the equine species. In study 2, hormone concentrations in the hair of mares hospitalized for attended parturition did not differ from those that were foaled at the breeding farm. This result could be related to a too brief period of hospitalization to cause significant changes in steroid deposition in the mare's hair.

Plant type I metacaspases are proteolytically active proteases despite their hydrophobic nature.

Plant metacaspases type I (MCA-Is), the closest structural homologs of caspases, are key proteases in stress-induced regulated cell death processes in plants. However, no plant MCA-Is have been characterized in vitro to date. Here, we show that only plant MCA-Is contain a highly hydrophobic loop within the C terminus of their p10 domain. When removed, soluble and proteolytically active plant MCA-Is can be designed and recombinantly produced. We show that the activity of MCA-I depends on calcium ions and that removal of the hydrophobic loop does not affect cleavage and covalent binding to its inhibitor SERPIN. This novel approach will finally allow the development of tools to detect and manipulate the activity of these cysteine proteases in vivo and in planta.

Adrenal Gland Ultrasonographic Measurements and Plasma Hormone Concentrations in Clinically Healthy Newborn Thoroughbred and Standardbred Foals.

Adrenal hormones, ACTH plasma concentrations and the ultrasonographic evaluation of the adrenal glands are considered complementary in clinical evaluations of the hypothalamic-pituitary-adrenal (HPA) axis function in several species. In dogs, age, size and weight have a significant effect on the ultrasonographic size of the adrenal glands. In neonatal foals, ultrasonographic evaluation and measurements of the adrenal glands have been demonstrated to be reliable; however, the effect of individual variables on ultrasonographic features has not been investigated, and the clinical usefulness of adrenal gland ultrasonography is still not known. The aims of this study were: (i) to provide and compare adrenal glands ultrasound measurements in healthy newborn Thoroughbred and Standardbred foals, and assess any effect of age, weight and sex on them; (ii) to assess and compare ACTH and steroid hormone concentrations in healthy neonatal foals of the two breeds. Venous blood samples and ultrasonographic images of the adrenal glands were collected from 10 healthy neonatal Thoroughbred and 10 healthy neonatal Standardbred foals. Ultrasonographic measurements of the adrenal glands were obtained and adrenocorticotropin (ACTH), cortisol, dehydroepiandrosterone (DHEA), progesterone (P4) and aldosterone plasma concentrations were assessed. The ACTH/cortisol, ACTH/progesterone, ACTH/aldosterone, ACTH/DHEA and cortisol/DHEA ratios were calculated. A significant positive correlation was found between the height of the right adrenal gland and the foal's weight; the width of the right and left adrenal gland and the medulla was significantly lower in Thoroughbreds than in Standardbreds. Cortisol and ACTH plasma concentrations were significantly higher and lower, respectively, in Thoroughbreds compared with Standardbreds. The cortisol/DHEA ratio was significantly higher in Thoroughbreds. This study provides reference ranges for neonatal Thoroughbred and Standardbred foals, and suggests the presence of breed-related differences in ultrasonographic adrenal gland measurements, plasmatic cortisol and ACTH concentrations. The higher cortisol/DHEA ratio detected in Thoroughbred foals could suggest a different response to environmental stimulation in the two breeds.

How Stressful Is Maternity? Study about Cortisol and Dehydroepiandrosterone-Sulfate Coat and Claws Concentrations in Female Dogs from Mating to 60 Days Post-Partum.

In dogs, the phase from mating to the end of weaning lasts about 120 days and encompasses many aspects that, interacting, contribute to increase the allostatic load. The coat and claws, useful for long-term change assessments, have the advantage of being collectable without invasiveness. In the present study, the Cortisol (C) and Dehydroepiandrosterone-sulfate (DHEA-S) concentration monthly changes in the coat and claws were studied in female dogs from mating to the end of weaning to assess Hypothalamic-Pituitary-Adrenal (HPA) axis activation during pregnancy and the post-partum period. The results from 15 Dobermann Pinscher female dogs showed a trend of increase of the coat C from mating to 60 days post-partum, with significant changes between mating and parturition-60 days post-partum (p < 0.01) and between the 30-day pregnancy diagnosis (PD) and 30-60 days post-partum (p < 0.05). The claws C trend showed significant increases between mating and 30-60 days post-partum (p < 0.05) and between the PD and 60 days post-partum (p < 0.01). DHEA-S in both matrices showed non-significant changes. The results suggest that maternity could play a pivotal role in the HPA axis activation, with a subsequent chronic secretion of C determining an increase in the allostatic load in the mothers. Neither maternal parity nor litter size played a significant role in the accumulation of C and DHEA-S in both matrices.

Effect of Delivery by Emergency or Elective Cesarean Section on Nitric Oxide Metabolites and Cortisol Amniotic Concentrations in at Term Normal Newborn Dogs: Preliminary Results.

The neonatal response to stress was reported to be related to the type of delivery, that is elective or emergency cesarean sections (ELCS and EMCS, respectively). Nitric oxide (NO) is also reported to be related to uterine inertia, and high levels of NO metabolites (NOs) are associated with physical and emotional stress. The study aimed to assess the concentrations of cortisol (C) and NOs in the amniotic fluid of puppies delivered by ELCS or EMCS. In total, 32 puppies were delivered by ELCS and 22 by EMCS. ANCOVA showed an effect of the ELCS vs. EMCS on both amniotic NOs (p < 0.001) and C (p < 0.001) concentrations. Lower amniotic C concentrations were found at increasing Apgar score (p < 0.001). Higher amniotic NOs concentrations were associated to increasing mother's parity (p < 0.001), puppies' birthweight (p < 0.001), and time of labor within the EMCS group (p < 0.05). A positive correlation between birthweight and amniotic NOs concentrations was also found (p < 0.05) in the EMCS group. Due to the possible concurrence of several compartments (maternal, maybe placental, and fetal) to the final amniotic fluid composition, the definition of the role played by the three compartments in the higher C and NOs concentrations found in amniotic fluids collected from puppies delivered by EMCS than ELCS needs further clarification.

Ultrasonographic measurement of the adrenal gland in neonatal foals: reliability of the technique and assessment of variation in healthy foals during the first five days of life.

BACKGROUND: Adrenal gland ultrasonographic measurements are useful in clinical evaluation of patients with adrenal dysfunction in several species. In human healthy neonates, the ultrasonographic size of the adrenal glands decreases during the first days of life. Ultrasonography of adrenal glands was demonstrated to be feasible in neonatal foals. The aims of this study were to describe a technique for ultrasonographic measurement of adrenal gland size to test its reliability in neonatal foals, and to assess any variation of ultrasonographic measurements during the first five days of life in healthy foals. METHODS: First, measurements of the adrenal glands were retrospectively obtained by three observers in 26 adrenal gland images of 13 healthy and sick neonatal foals. The interobserver and intraobserver agreement were tested. Later, adrenal gland ultrasonographic images and measurements were acquired by one operator in 11 healthy neonatal foals at one, three and five days of life and differences among the measurements obtained at the different time points were assessed. RESULTS: Interobserver agreement ranged from fair to excellent (0.48-0.92), except for cortex width (<0.4); intraobserver agreement ranged from good to excellent (0.52-0.98). No significant differences were found among the measurements obtained at one, three and five days of life. CONCLUSION: Adrenal glands ultrasonographic measurements can be obtained consistently in equine neonates, and in contrast to people they do not vary during the first five days of life in healthy foals.

Determination of Fatty Acids Profile in Original Brown Cows Dairy Products and Relationship with Alpine Pasture Farming System.

This study aimed to evaluate the relationships between fatty acids and the pattern that most contributes to discriminate between two farming systems, in which the main difference was the practice, or not, of alpine summer-grazing. Milk and cheese were sampled every month in two farms of Original Brown cows identical under geographical location and management during no grazing season point of view in the 2018 season. Fatty acids concentrations were determined by gas chromatography. The principal component analysis extracted three components (PCs). Mammary gland de novo synthetized fatty acids (C14:0, C14:1 n9, and C16:0) and saturated and monosaturated C18 fatty acids (C18:0, C18:1 n9c) were inversely associated in the PC1; PC2 included polyunsaturated C18 fatty acids (C18:2 n6c, C18:3 n3) and C15:0 while conjugated linoleic acid (CLA n9c, n11t) and fatty acids containing 20 or more carbon atoms (C21:0, C20:5 n3) were associated in the PC3. The processes of rumen fermentation and de novo synthesis in mammary gland that are, in turn, influenced by diet, could explain the relationships between fatty acids within each PC. The discriminant analyses showed that the PC2 included the fatty acids profile that best discriminated between the two farming systems, followed by PC3 and, lastly, PC1. This model, if validated, could be an important tool to the dairy industry.

Steroids in pig hair and welfare evaluation systems: combined approaches to improve management in pig breeding?

The aim of the present pilot study was to determine the allostatic load by analysing the hair cortisol and dehydroepiandrosterone­sulphate (DHEA­S) of commercial pigs belonging to different farms having good overall animal welfare according to the CREnBA (Centro di Referenza Nazionale per il Benessere Animale - Brescia, Italy) assessment protocol. The study was conducted on 86 pigs belonging to three farms with a grade higher than 8 on the CReNBA welfare evaluation system. Hair samples were taken by shaving on sows 1­10 days after weaning (Farms 1 and 2) and at the age of 8­10 months (Farm 3). The hormone concentrations were measured by radioimmunoassay. From the box plots, it is evident that the hair cortisol concentrations of animals were different between farms. Conversely, the variability of the hair DHEA­S concentration was similar between the F1 and F2 farms but much lower at F3. For all the variables considered (cortisol, DHEA­S and cortisol/DHEA­S ratio), F2 showed a higher hair concentration level than F1 (P < 0.05). The study's results indicate that the measurement of cortisol and DHEA­S in pig hair shows a different allostatic load between them, although the official welfare evaluation method assessed the farms as having good overall animal welfare.

Effects of Alfaxalone or Propofol on Giant-Breed Dog Neonates Viability During Elective Caesarean Sections.

Attention must be paid to C-section anesthesia effects on mother and offspring. Alfaxalone induction results in improved puppy viability when compared to propofol. The present study aims to evaluate effects of alfaxalone or propofol induction for elective C-section on newborns, expressed as Apgar score and fetal fluids cortisol concentration. Anesthesia was induced with alfaxalone 3 mg/kg iv in 5 bitches (group A), and propofol 4 mg/kg iv in another 5 (group P), maintained with isoflurane. Amniotic and allantoic fluids were collected to determine cortisol concentration. Apgar score, litter size, newborn gender, birth-weight, maternal age, and parity were recorded. ANOVA, U Mann-Whitney test and ANCOVA assessed the effects of drugs on the Apgar score and fetal fluids cortisol. Thirty-six puppies were randomly selected for the study: 16 from group A and 20 from group P. Only the Apgar score significantly differed between groups. ANCOVA confirmed a significantly higher Apgar score in group A underlining the influence of fetal fluids cortisol concentrations, both resulting in covariates. Present results confirm the effect of anesthesia on the Apgar score of newborns, which is significantly higher for alfaxalone than propofol, suggesting the use of fetal fluids cortisol as a covariate. These findings could be a starting point for further investigations when less viable puppies are detected or expected, such as during an emergency C-section.

Analysis of 19 Minerals and Cortisol in Red Deer Hair in Two Different Areas of the Stelvio National Park: A Preliminary Study.

The aim of the study was to perform an investigation on the concentration of 19 minerals and cortisol in red deer (Cervus elaphus) hair, a matrix that is easy to collect with non-invasive and painless sampling, able to represent an integrative values of long-term substance concentrations, and able to give useful information, also when performed on dead animals, given its extreme stability over time. In the study thirty-five animals were included, coming from two different sides of a valley in the Stelvio National Park, where official water analysis had pointed out elevated concentrations of As in one of the two orographic sides. Hair cortisol concentrations were measured using a RIA(Radio Immuno Assay), while minerals were detected using ICP-MS (Inductively Coupled Plasma- Mass Spectrometry). Results showed a negative relationship between cortisol and some mineral concentrations (Li, Co, As, Cd, Cr and Tl) and significant differences in some mineral concentrations between park areas (Al, Co, Cu, Cd and Ni). As, Cr and cortisol differences approached statistical significance. This preliminary study represents a step forward in the study of wildlife allostatic load and a valid method for applications in wildlife management programs, in environmental studies and in public health programs.

Cortisol, DHEA, and Sexual Steroid Concentrations in Fattening Pigs' Hair.

The aim of this study was to analyze the feasibility and reliability of using hair as a matrix to determine the dehydroepiandrosterone (DHEA) and sexual steroid concentrations and the cortisol/DHEA ratio in fattening pigs. The results could be also used to plan future research to identify threshold values in order to set up strategies to control the allostatic load and increase the resilience of fattening pigs before slaughter. The study was conducted on 107 commercial crossbred rearing pigs. The hair samples were taken by shaving at the age of 36 weeks, and concentrations of the hormones were measured using a solid-phase microtiter radioimmunoassay. Females had significantly higher cortisol levels (p < 0.01), significantly lower DHEA concentrations (p < 0.05) and significantly higher cortisol/DHEA ratios (p < 0.01) than barrows. Progesterone was significantly higher in gilts than in barrows (p < 0.01). Testosterone and 17ß-estradiol were significantly higher in barrows than in gilts (p < 0.05). If future research can produce threshold values for the different markers examined, the evaluation of animals under subclinical stress conditions will be possible.

Relocation and Hair Cortisol Concentrations in New Zealand White Rabbits.

To investigate how long relocation modified hair cortisol concentrations in New Zealand white rabbits, 19 rabbits were subjected to a change in their breeding facility at the beginning of the trial and then were kept under stable environmental conditions. Hair samples were collected at the time of arrival to the nonhuman animal facility and at 40-day intervals from the same skin area for up to 440 days after the animals' arrival to the facility. A period effect on the hair cortisol concentration was found (p < .01). The transfer of the rabbits to the new facility might have induced an increase in the hypothalamic-pituitary-adrenal axis activity (p < .01). A second increase in hair cortisol concentration (p < .01) occurred at 320 days, after a change of personnel at the facility that occurred at 280 days, which was the only environmental change. The relocation of rabbits to the facility resulted in a stress response leading to elevated cortisol levels. The effect of relocation on mean cortisol concentrations was exhausted within 120 days when all environmental factors were kept stable.