Cortisol Metabolites Research Articles

Don't forget the dams! Dairy cows' responses to two separation methods after 3 months of cow-calf contact

Weaning and separation remain among some of the biggest challenges for cow-calf contact systems, making the development of practical and low stress separation methods mandatory for future success of these systems. This study aimed to compare behavioral and physiological responses of dairy cows to separation from their calves after 3 mo of full-time contact with either the 2-step method (NF, 2 wks full-time contact while calves wore a nose flap, 1 wk fence-line contact before total separation, n = 18) or by gradual reduction of contact time between cow and calf (GR, 1 wk half day contact, 1 wk morning contact, 1 wk fence-line contact before total separation, n = 18). Vocalizations and searching behavior were observed on 4 d/wk from 1 wk before separation until 1 wk after total separation. During the same period, lying behavior and rumination time was automatically assessed via accelerometers and pressure sensors. Fecal and blood samples were collected twice/wk from day -1 till +23 relative to separation start for analysis of fecal cortisol metabolites and the immune response. Milk yield in the parlor was continuously recorded. Statistical analysis was conducted using linear mixed effects models. We found no difference between the 2 separation methods in any of the examined behavioral and physiological response variables. However, a significant increase in vocalizations and searching behavior compared with baseline was present with both methods. Further, there was a transient increase in physiological distress markers and a short-lived retention of milk yield at initiation of treatments, indicating that both methods induced distress for the cows. Descriptively, there were large inter-individual differences between cows as well as a different temporal distribution in occurrence of behavioral responses, as GR cows vocalized most frequently during the week with fence-line contact and after total separation from the calf, while NF cows reacted strongest during the 2 wks while calves wore the nose flap. Milk yield was higher in NF than GR cows during the 2 wks while GR calves had time-restricted access to their dams and NF calves were prevented from suckling, but showed no difference afterward anymore. However, similar evening milk yields of GR cows in the weeks with half day and morning contact, indicated that the weekly reductions in contact time worked in a rather stepwise than gradual manner, which warrants further improvement of the GR method. Taken together, results showed that cows experienced distress during separation with the GR method, when implemented over 3 wks in 3 steps, as well as with the NF method, but differences between individuals were considerable.

12430 Urine Steroid Profiling In Patients With Unilateral And Bilateral Mild Autonomous Cortisol Secretion

Abstract Disclosure: B. Salama: None. J. Saini: None. V. Fell: None. E.J. Atkinson: None. S.J. Achenbach: None. Background: Mildly autonomous cortisol secretion (MACS) can be unilateral in 80% of cases, or bilateral in 20% of cases, due to either bilateral adrenal adenomas or bilateral macronodular adrenal hyperplasia. Scarce evidence demonstrates the differences in plasma steroid profiling of patients with bilateral versus unilateral MACS, however, these findings have not been confirmed using a more comprehensive 24h urine steroid profiling. Objectives: To characterize 24h urine steroid metabolome in patients with unilateral and bilateral MACS. Methods: We conducted a single-center cross-sectional study between 2019-2022. Eligible participants included consecutive patients with MACS who provided a 24h urine sample. Urine was analyzed for 25 steroids with a high-resolution mass spectrometry assay. MACS was defined as cortisol >1.8 mcg/dL following the overnight administration of 1 mg dexamethasone (DST-cortisol). Partial cohort analysis is presented. Results: Of 72 patients with MACS included in the interim analysis, 36 (50%) had bilateral MACS. The two groups (unilateral vs bilateral) were similar in age (median 58 vs 58 years), sex (women 67% vs 61%), BMI (median 33 vs 32 kg/m2), and menopausal status (36% vs 39%), P>0.05 for all. Prevalence of comorbidities was also similar in both groups: hypertension (89% vs 89%), hyperlipidemia (69% vs 78%), and hyperglycemia (69% vs 61%), P >0.05 for all. While the DST-cortisol (median 3.0 vs 3.2 mcg/dL, P= 0.51) and dehydroepiandrosterone sulfate (DHEAS) (median 37.5 vs 55.0, P = 0.21) were similar between the 2 groups, patients with bilateral MACS had a lower ACTH (median 12.4 vs 8.2 pg/mL, P = 0.012) when compared to patients with unilateral MACS. Median adrenal mass size was similar in patients with both unilateral MACS (2.7 cm, IQR 1.7-3.9) and bilateral MACS (3.0 cm, IQR 2.2-3.6) P = 0.54. Of the 25 steroids measured, only tetrahydrocorticosterone was higher in patients with unilateral MACS (median 245 mcg/24h, IQR 165-416 vs median 184 mcg/24h, IQR 121 -278) P = 0.039. No differences in androgens (median 2229 vs 2036 mcg/24h), cortisol metabolites (median 6566 vs 5191 mcg/24h), cortisone metabolites (median 10300 vs 9429 mcg/24h), or glucocorticoid/androgen ratio (median 7.9 vs 8.9) was demonstrated between the unilateral vs bilateral MACS groups (P>0.05 for all). Conclusion: In this interim analysis of patients with unilateral vs bilateral MACS, we have found that patients had a similar post-DST cortisol and similar tumor bulk. Despite a lower ACTH in patients with bilateral MACS, no differences in androgens were found between the two groups. In addition to expanding steroid profiling analysis to the whole cohort, subgroup analyses will need to distinguish between patients with bilateral adenomas and macronodular adrenal hyperplasia, consider aldosterone co-secretion, and employ a more accurate measurement of adrenal tumor bulk. Presentation: 6/3/2024

Polymorphism in the Drug Transporter Gene ABCB1 as a Potential Disease Modifier in Cortisol-Producing Adrenal Adenomas.

Endogenous hypercortisolism presents with variable phenotypes. Etiological factors accounting for the level of hypercortisolism or varying severity of associated comorbidities are lacking. Recently, the adrenal ATP-binding cassette B1 (ABCB1) gene was identified as a modulator of glucocorticoid secretion. To evaluate the effect of ABCB1 polymorphism rs2032582 on steroid metabolome and clinical phenotypes in patients with endogenous hypercortisolism. In this cross-sectional cohort study, 137 patients prospectively enrolled in the German Cushing's registry were included (41 with ACTH-producing pituitary adenoma, 21 with cortisol-producing adrenal adenoma, and 75 with excluded hypercortisolism). In all patients, ABCB1 polymorphism was analyzed using a TaqMan genotyping assay, glucocorticoid metabolite excretion in 24-hour urine samples was analyzed by gas chromatography-mass spectrometry, and the clinical phenotype was assessed systematically. In patients with cortisol-producing adrenal adenomas, but not in patients with ACTH-producing pituitary adenomas, homozygous major allele GG of ABCB1 polymorphism rs2032582 was associated with higher overall cortisol metabolite secretion (median 13515 [IQR 10347; 25669] µg/24h vs. 9645 [6146; 10732] µg/24h in minor homo- and heterozygotes, p=0.036) and elevated major cortisol metabolites αTHF, THF and THE (9339 [6929; 17789] µg/24h vs. 6288 [4184; 7455] µg/24h, p=0.045). Moreover, these patients showed higher mean arterial pressure (116 [111; 131] mmHg in major homozygotes vs. 105 [96; 112] mmHg in minor homo- and heterozygotes, p=0.036). The genotype of drug transporter gene ABCB1 rs2032582 polymorphism is associated with the degree of cortisol metabolite secretion in cortisol-producing adrenal adenomas and could, therefore, represent a modifier of disease severity in this context.

Relationships between boldness and fecal steroid hormone levels in captive alpine musk deer (Moschus chrysogaster) in China

Animals adapt to environmental changes through behavioral and physiological regulation, and there are individual differences in their ability to adapt to the environment, which are associated with individual personality. Boldness is a fundamental axis of animal personality, characterizing an animal's response to unfamiliar environments and threats. From June 1 to July 28, 2023, a personality assessment experiment utilizing a novel stimulus test with an unfamiliar human was conducted on 39 captive alpine musk deer (Moschus chrysogaster) at the Zhuanglang Musk Farm in Gansu Province, China. We recorded avoidance reaction indicators of musk deer in the presence of an unfamiliar human, and measured the level of the musk deer's boldness, influencing factors, and the relationships with fecal steroid hormone levels. Based on cluster analysis, 39 captive musk deer could be divided into bold (boldness score, BSS = 0.994 ± 0.008, n = 3) and shy groups (BSS = 0.327 ± 0.145, n = 36) and the average BSS in 39 musk deer was 0.378 ± 0.228. Factors such as age, sex, and density of enclosures did not have a significant effect on the boldness of musk deer. There was a significant negative correlation between the musk deer's boldness and the level of fecal cortisol metabolites. However, the correlations between boldness and testosterone levels in males and estradiol levels in females were not significant. Based on the results of our study, in the practice of musk deer farming, the measurement of the boldness can help predict the individual's adaptation to environmental stress, and it can also provide a new research perspective for the study of the boldness of solitary animals that are forced to live in groups.

A randomized controlled trial to study the effect of supplemental premilking stimulation on milking performance, teat tissue condition, udder health, and well-being in dairy cows

The objectives of this study were to evaluate the effect of supplemental premilking stimulation, provided after manual stimulation, by means of high-frequency pulsation without reduction of the vacuum in the pulsation chamber on milking performance, teat tissue condition, udder health, and well-being in dairy cows. In a randomized controlled trial, Holstein cows (n = 491) from 1 commercial dairy farm with a thrice-per-day milking schedule were assigned to treatment and control groups over a 64-d period. Treatments consisted of a maximum of 20 s of pulsation stimulation at a pulsation rate of 100 (SPS100) or 300 (SPS300) cycles per minute and a pulsation ratio of 25:75, which were applied after completion of manual premilking stimulation upon milking unit attachment. Cows in the control group (CON) received only traditional premilking stimulation by manual forestripping for 6 s, and wiping. Milking characteristics were measured with on-farm milk flow meters. Milking machine-induced short-term (swelling at teat base, firmness at teat end, and teat discoloration) and long-term (teat-end callosity) changes to the teat tissue were assessed manually and visually. Composite milk samples were analyzed for somatic cell counts. Cow hind-leg activity was assessed with 3-dimensional accelerometers. Fecal 11,17-dioxoandrostanes (11,17-DOAs; a group of cortisol metabolites) were determined on wk 4 and 8 to assess the well-being of the cows. Generalized linear mixed models were used to study the effect of treatments on the outcome variables milk yield/milking and milking unit-on time. We observed no meaningful differences among groups for milk yield or milking unit-on time. Least squares means and their 95% confidence intervals (95% CI) for cows in the SPS100, SPS300, and CON groups were 13.9 (13.3-14.5), 14.0 (13.4-14.6), and 13.9 (13.3-14.6) kg for milk yield and 218 (212-224), 218 (211-224), and 218 (212-224) s for milking unit-on time, respectively. Compared with cows in the CON group, the odds (95% CI) of short-term changes were 1.30 (0.95-1.78) for the SPS100 group and 1.50 (1.10-2.05) for the SPS300 group. The odds of long-term changes were 0.94 (0.67-1.34) for cows in the SPS100 group and 0.71 (0.49-1.04) for cows in the SPS300 group. We observed no differences in SCC. In reference to the CON group, the hazard ratio (95% CI) in SPS100 and SPS300, respectively, were 0.35 (0.13-0.98) and 1.22 (0.57-2.64) for clinical mastitis, and 0.34 (0.12-0.95) and 1.28 (0.60-2.73) for culling. The least squares means (95% CI) of hind-leg activity during milking were 8.3 (6.5-10.5), 10.6 (8.1-13.7), and 9.1 (7.2-11.6) movements per milking for the SPS100, SPS300, and CON groups, respectively. The least squares means (95% CI) of fecal 11,17-DOAs concentration (ng/g) at the first and second test days, respectively, were 31.1 (28.1-34.2) and 22.3 (19.2-25.4) for the SPS100 group, 26.4 (23.4-29.4) and 25.2 (22.0-28.4) for the SPS300 group, and 24.8 (21.8-27.9) and 25.0 (21.7-28.3) for the CON group. We conclude that applying supplemental stimulation after manual stimulation through the high-frequency pulsation system tested here did not impart additional benefits to the milk harvesting process, teat tissue condition, somatic cell count, or the well-being of the animals.

Hibernating or not hibernating? Brown bears' response to a mismatch between environmental natural cues and captive management, and its welfare implications.

In wild brown bears, likely factors triggering hibernation response to harsh environmental conditions are temperature, photoperiod, and food resources availability. In fact, constantly fed captive brown bears are described as skipping hibernation being active all year-round. Is the hibernation response so flexible and subordinate to contingencies, or else is an adaptation that, if dismissed, may negatively impact on bear well-being? This study investigates the potential hibernation response in captive brown bears under unvaried management conditions using an integrative approach simultaneously analyzing multiple animal-based variables together with environmental covariates. Data from a mid-latitude zoo revealed distinct behavioral, fecal glucocorticoids, and body condition score seasonal fluctuations, resembling natural hibernation cycles, despite constant food access. Environmental variables like photoperiod and visitor numbers significantly influenced activity levels. Bears exhibited behaviors indicative of hyperphagia and fall transition, such as appetitive feeding and denning behaviors. Hormonal analyses revealed high fecal cortisol metabolites levels during hyperphagia, suggesting physiological responses to seasonal changes. Findings underscore the importance of environmental cues and food availability in shaping zoo bear behavior and physiology. Considering that the hibernating vs. non-hibernating description might represent an oversimplification, management strategies should deal with captive bear potential need to freely express their adaptive predispositions by accommodating their natural behaviors, such as providing denning spots and adjusting diet composition as soon as typical hyperphagic and predenning behaviors emerge, ultimately enhancing their well-being.

Investigating the biological relevance of measuring gastrointestinal cortisol metabolite levels to assess stress responses in Atlantic salmon (Salmo salar L.) after an acute stress

Monitoring stress levels of farmed Atlantic salmon (Salmo salar) is important to ensure fish welfare and optimize farm operations. Feces could be a promising matrix for assessing stress responses in fish, based on their properties of low-invasive sampling and allowing repeated sampling over time. Meanwhile, elevated levels of cortisol metabolites (CMs) in feces indicate the increases in plasma cortisol levels (PLA) after exposure to acute stress. However, the dynamics of fecal CMs following acute stress in Atlantic salmon remain unclear. In this study, a confinement stress involving chasing and crowding was conducted to investigate the responses of gastrointestinal CMs to an acute stressor in Atlantic salmon. The post-smolts, with an average weight of 155.21 g, were sampled before and at 30 min, 1.5, 6, 12, 18, 24, 36, and 48 h after the onset of stress. Blood and gastrointestinal contents from the stomach, proximal intestine, and distal intestine of each fish were collected and subsequently analyzed, using competitive enzyme-linked immunosorbent assay (ELISA). The results demonstrated that the pre-stress level of PLA was low (4.28 ± 6.13 ng/ml) and reached a peak within 30 min following stress. The levels of CMs in gastrointestinal contents from stomach (SCMs), proximal intestine (PCMs), and distal intestine (DCMs) in pre-stress group were 0.82 ± 0.50, 18.31 ± 6.14 and 16.04 ± 6.69 ng/g, respectively. Gastrointestinal CMs increased significantly within 30 min and the peak levels of SCMs (3.51 ± 3.75 ng/g), PCMs (68.19 ± 23.71 ng/g) and DCMs (65.67 ± 23.37 ng/g) were found at 1.5 h post-stress. The significant increases in PCMs and DCMs post-stress validate the biological relevance of measuring intestinal CMs for assessing acute stress responses in Atlantic salmon. No significant difference was noted between PCMs and DCMs across all samples, suggesting that intestinal contents can serve as a suitable matrix compared with feces when measuring the responses of CMs to acute stress. The time lag between the peak of PLA levels and their reflection in the intestinal contents exceeded 1 h, indicating that using intestinal contents as a matrix to assess stress levels in fish can extend and delay the sampling window. This study highlights valuable guidance for determining the optimal times to utilize intestinal contents for measuring stress responses, providing further insights into the dynamics of fecal CM following acute stress.

Sampling time for different matrices in stress assessment of farmed Atlantic salmon post-smolt

The sustainability of commercial aquaculture production depends critically on prioritizing fish welfare management. Besides monitoring welfare parameters such as fish behaviour and water quality, fish stress level can also provide a reliable measure of the welfare status of farmed fish. Cortisol and 5 of its metabolites (5β-THF, cortisone, 5β-DHE, 5β-THE, β-cortolone) were previously identified by the authors as suitable stress biomarkers of farmed Atlantic salmon. Based on this knowledge, the present study aimed to investigate the time-related dynamics of these metabolites in plasma, skin mucus, bile and faeces over a 72 h- period. The objective was to determine the optimal sampling time for each matrix and to understand the clearance pathway of these metabolites following stress. An experiment was carried out using a total of 90 Atlantic salmon with an average weight of 438 (±132) g. The average sea temperature was 6.9 °C during the experimental period. A control group of 10 fish was first collected before the remaining 80 fish were submitted to a stress of netting and subsequent relocation into two separate cages. From each of these two stress groups, 10 fish were sampled at 1 h, 2 h, 4 h, 6 h and 12 h, 24 h, 48 h, 72 h after the stress event respectively. The concentrations of cortisol and its metabolites were measured at each of the sampling timepoint. The results demonstrated that plasma cortisol metabolites reached the highest concentration 4 h after stress and remained elevated despite the slight decrease for the remaining timepoints. The peak level was observed at 12 h post-stress in skin mucus and 24 h in bile and faeces. The findings suggest that these timepoints are the optimal for sampling Atlantic salmon post-smolt following stressful events in acute stress studies. Furthermore, the results reveal that analysing cortisol and its metabolites, both in free and conjugated forms, rather than free cortisol provides greater flexibility as their concentrations are less affected by sampling procedure. This study confirms the appropriateness of skin mucus and faeces as less-invasive sample matrices for fish stress evaluation and provides a basis for further developing low invasive tools for monitoring the welfare of farmed salmonid.

LC-HRMS and GC-MS Profiling of Urine Free Cortisol, Cortisone, 6Β-, and 18-Hydroxycortisol for the Evaluation of Glucocorticoid and Mineralocorticoid Disorders.

Urine free cortisol measurements are routinely performed to evaluate hypercortisolism. Despite their analytical inaccuracy, immunoassay-based methods are frequently used. Advances in liquid chromatography-high-resolution mass spectrometry (LC-HRMS) facilitate the incorporation of powerful diagnostic tools into clinical laboratories. In addition to its high analytical specificity and simultaneous analysis of different metabolites, accurate mass measurement allows for untargeted compound identification, which may help to identify clinically relevant metabolites or drugs. The present study aimed to validate a simple routine LC-HRMS method to quantify cortisol, cortisone, 6β-hydroxycortisol, and 18-hydroxycortisol simultaneously in human urine. Additionally, the study also validated a GC-MS method for the same steroids, evaluated their cross-reactivity with commercial cortisol immunoassays, and quantified the 24 h urine excretion in patients under clinical suspicion or follow-up for hypercortisolism. The LC-HRMS method involved liquid-liquid extraction using dichloromethane, micro-LC for chromatographic separation and detection using the accurate masses of the steroids, and simultaneous high-resolution full scan acquisition. The method presented acceptable linearity, precision, and accuracy. Significant interference from 6β-hydroxycortisol and cortisone was demonstrated in the cortisol immunoassays, which impacted their reliability in the follow-up of patients with hypercortisolism and significant changes in these cortisol metabolites (i.e., due to drug-induced changes in CYP3A4 activity). A rapid and accurate routine LC-HRMS method was validated, which is useful for the evaluation of hypercortisolism and other disorders of glucocorticoid and mineralocorticoid metabolism.

Administration of chromium picolinate and meloxicam alleviates regrouping stress in dairy heifers.

This research investigated the effect of administering chromium (Cr) and meloxicam (MEL) on growth performance, cortisol and blood metabolite, and behaviors in young, regrouped heifers. Fifty Holstein dairy heifers (body weight [BW] 198±32.7 kg and 6.5±0.82 months of age) were randomly assigned to non-regrouped group or four regrouped groups. Non-regrouped animals were held in the same pen throughout the entire experimental period (NL: non-regrouping and administration of lactose monohydrate [LM; placebo]). For regrouping groups, two or three heifers maintained in four different pens for 2 weeks were regrouped into a new pen and assigned to one of four groups: regrouping and LM administration (RL); regrouping and Cr administration (RC); regrouping and MEL administration (RM), and regrouping and Cr and MEL administration (RCM). LM (1 mg/kg BW), Cr (0.5 mg Cr picolinate/kg dry matter intake), and MEL (1 mg/kg BW) were orally administered immediately before regrouping. Blood was collected before regrouping (0 h) and at 3, 9, and 24 h and 7 and 14 d thereafter. Behaviors were recorded for 7 consecutive days after regrouping. Average daily gain was lower (p<0.05) in RL than NL heifers, but was higher (p<0.05) in RM, RC, and RCM than RL heifers. RL heifers had higher (p<0.05) cortisol than NL heifers on d 1 after regrouping. The cortisol concentrations in RC, RM, and RCM groups were lower (p<0.05) than in RL treatment 1 d after regrouping. Displacement behavior was greater (p<0.05) in RL group than all other groups at 2, 3, and 6 d after regrouping. Regrouping caused temporal stress, reduced growth performance, and increased displacement behavior in heifers. Administering Cr and MEL recovered the retarded growth rate and reduced displacement behavior, thereby alleviating regrouping stress.

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.

Environmental enrichment during yard weaning alters the performance of calves in an attention bias and a novel object recognition test

The weaning of beef calves in yards places multiple stressors on the animals, and environmental enrichment may help mitigate some of these stressors and improve animal welfare. This trial assessed the impacts of enrichment provision to beef calves during yard weaning using measures of biological functioning, behaviour, and affective state. Overall, calves utilised the brush more than the other provided enrichments, which were a hanging rope and a ball. Enrichment influenced the behaviours of calves during both an attention bias test, with enriched calves exhibiting behaviours associated with greater anxiety, and a novel object recognition test, with enriched calves spending less time interacting with objects. In their home pens, enriched calves performed more drinking and grooming behaviours. However, no significant differences were seen between treatments for body weight, faecal cortisol metabolites, and internal body temperature. Enrichment also did not influence any longer-term measurements of body weight, flight speed, or crush score. The study design was impacted by mud, requiring the regrouping of the animals. Thus, some results should be interpreted with caution. Overall, this study demonstrates that further work is required into the assessment of affective states for these animals as it could not be confirmed whether the results seen indicate that calf welfare was improved or impaired through enrichment provision.

The impact of net cleaning on fecal cortisol metabolites in farmed Atlantic Salmon

Stress assessment is crucial in aquaculture to ensure and improve the health and well-being of production animals including fish. The primary objective of this study was to utilize the assessment of fecal cortisol metabolites (FCMs) as a low-invasive stress determination method for farmed Atlantic salmon (Salmo salar L.) and to suggest an appropriate sampling timeframe for this measurement.Blood, mid-intestinal contents, and feces samples were collected before, during, and at 2 and 20 h after a stress-inducing event, namely, net cleaning. Plasma cortisol (PC) and its metabolite levels in the mid-intestinal contents (MICMs) and FCMs were assessed by an enzyme-linked immunosorbent assay (ELISA). The research reveals a distinctive temporal profile, with a sequential elevation in levels of PC, MICMs and FCMs in response to the effects of net cleaning. Peak FCMs levels were observed approximately 20 h after net cleaning and were significantly higher than peak PC levels (P < 0.05). The findings suggest that FCMs are a promising indicator for acute stress assessment. To enhance the understanding and improve the applicability of FCMs as stress indicators in fish farming practices the critical challenges regarding feces sampling and FCMs analyses are further discussed. Future research aimed at developing practical protocols and industry standards will emphasize method validation and include a greater number of sampling points as well as an expanded experimental period.This study signifies a crucial step toward understanding the role of FCMs as a reliable stress indicator in fish. It underscores the importance of accounting for the delayed and extended sampling patterns when assessing stress in aquatic settings. Moreover, it offers a less invasive and more labor-efficient approach for monitoring the overall health and welfare of salmonids than PC analysis, thereby promoting sustainability in this vital industry.

Steroid Metabolomic Signature in Term and Preterm Infants.

Adrenal function is essential for survival and well-being of preterm babies. In addition to glucocorticoids, it has been hypothesized that C19-steroids (DHEA-metabolites) from the fetal zone of the adrenal gland may play a role as endogenous neuroprotective steroids. In 39 term-born (≥37 weeks gestational age), 42 preterm (30-36 weeks) and 51 early preterm (<30 weeks) infants 38 steroid metabolites were quantified by GC-MS in 24-h urinary samples. In each gestational age group, three distinctive cluster were identified by pattern analysis (k-means clustering). Individual steroidal fingerprints and clinical phenotype were analyzed at the 3rd day of life. Overall, the excretion rates of C21-steroids (glucocorticoid precursors, cortisol, and cortisone metabolites) were low (<99 μg/kg body weight/d) whereas the excretion rates of C19-steroids were up to 10 times higher. There was a shift to higher excretion rates of C19-steroids in both preterm groups compared to term infants but only minor differences in the distribution of C21-steroids. Comparable metabolic patterns were found between gestational age groups: Cluster 1 showed mild elevation of C21- and C19-steroids with the highest incidence of neonatal morbidities in term and severe intraventricular hemorrhage in early preterm infants. In cluster 2 lowest excretion in general was noted but no clinically unique phenotype. Cluster 3 showed highest elevation of C21-steroids and C19-steroids but no clinically unique phenotype. Significant differences in steroid metabolism between clusters are only partly reflected by gestational age and disease severity. In early preterm infants, higher excretion rates of glucocorticoids and their precursors were associated with severe cerebral hemorrhage. High excretion rates of C19-steroids in preterm infants may indicate a biological significance.

Management of dairy heifers: Can operant conditioning be an effective and feasible tool to decrease stress and ease animals' close contact and handling?

Beside health monitoring, a regular check of dairy heifers' growth rate is desirable, but it is rarely done since procedures that require restraint and handling can be associated with substantial stress for both animals and farmers. Inexperienced heifers, especially if they are highly responsive to humans, may find restraint and handling potentially aversive. This study investigated whether training heifers of different age and responsiveness toward humans (RTH), through operant conditioning, could reduce stress in animals, ease close contact and handling, and be feasible in terms of farmer's effort. We assessed 60 Holstein heifers of 2 age classes (Young, n = 29, 291 ± 39; Old, n = 31, 346 ± 62 d) according to the Avoidance Distance Test (ADT) and classified them as Confident (C, n = 20), Neutral, (N, n = 21) or Non-confident (NC, n = 19). Half of the heifers of each age and RTH class was trained (Tr, n = 29), whereas the other half was not (NTr, n = 31). The Tr heifers were subjected to target training for 8 sessions and positively reinforced with feed to allow being touched on the muzzle, rump and perineum. In case a heifer refused positive reinforcement, the trainer stepped back as negative reinforcement. In the last week of the experiment the effect of training on the reaction to handling was assessed in all heifers. We measured heart rate (HR), root mean square of successive inter-beat interval differences (RMSSD) and fecal cortisol metabolites (FCMs). The presence of behavioral distress signs was recorded as well. The ADT was performed a second time 24 h after the measuring session. All the trained heifers, regardless of RTH class, successfully accomplished the target training task in 6 sessions, spending on average 25.3 s per session, each. All the trained heifers allowed to be touched on the rump and perineum at the end of the 4th session. Training NC heifers required more time compared with the others. Trained heifers showed higher RMSSD (14.2 vs. 16.9), indicating a lower vagal tone, and thus, a slightly lower stress level than NTR. Training did not lead to differences in HR, FCMs or presence of stress behavioral signs. NC heifers had the highest mean baseline FCM values (38.4 vs. 30.3 vs. 29.1 ng/g) compared with N and C. NC heifers showed also the lowest value of FCMs, 12 h after the measuring session (36.7 vs. 44.6 vs. 49.7 ng/g), likely due to a decreased responsiveness of the adrenal gland to a stressor. The average avoidance distance decreased between the beginning and the end of the experiment, especially for N and NC heifers, regardless whether they were trained or not. These results show how using operant conditioning on some heifers, not only decreased their vagal tone, but reduced the responsiveness to humans of all the animals, trained and not trained; in the latter case, through non-associative learning, such as habituation.

Extensive comparison of salivary collection, transportation, preparation, and storage methods: a systematic review

BackgroundHuman saliva as a bodily fluid—similar to blood—is utilized for diagnostic purposes. Unlike blood sampling, collecting saliva is non-invasive, inexpensive, and readily accessible. There are no previously published systematic reviews regarding different collection, transportation, preparation, and storage methods for human saliva.DesignThis study has been prepared and organized according to the preferred reporting items for systematic reviews and meta-analyses (PRISMA) 2020 guidelines. This systematic review has been registered at PROSPERO (Registration ID: CRD42023415384). The study question according to the PICO format was as followed: Comparison of the performance (C) of different saliva sampling, handling, transportation, and storage techniques and methods (I) assessed for analyzing stimulated or unstimulated human saliva (P and O). An electronic search was executed in Scopus, Google Scholar, and PubMed.ResultsTwenty-three descriptive human clinical studies published between 1995 and 2022 were included. Eight categories of salivary features and biomarkers were investigated (i.e., salivary flow rate, total saliva quantity, total protein, cortisol, testosterone, DNA quality and quantity, pH and buffering pH). Twenty-two saliva sampling methods/devices were utilized. Passive drooling, Salivette®, and spitting were the most utilized methods. Sampling times with optimum capabilities for cortisol, iodine, and oral cancer metabolites are suggested to be 7:30 AM to 9:00 AM, 10:30 AM to 11:00 AM, and 14:00 PM to 20:00 PM, respectively. There were 6 storage methods. Centrifuging samples and storing them at -70 °C to -80 °C was the most utilized storage method. For DNA quantity and quality, analyzing samples immediately after collection without centrifuging or storage, outperformed centrifuging samples and storing them at -70 °C to -80 °C. Non-coated Salivette® was the most successful method/device for analyzing salivary flow rate.ConclusionIt is highly suggested that scientists take aid from the reported categorized outcomes, and design their study questions based on the current voids for each method/device.

Profiling endogenous adrenal function during veno-venous ECMO support in COVID-19 ARDS: a descriptive analysis.

Prolonged critical illness is often accompanied by an impairment of adrenal function, which has been frequently related to conditions complicating patient management. The presumed connection between hypoxia and the pathogenesis of this critical- illness- related corticosteroid insufficiency (CIRCI) might play an important role in patients with severe acute respiratory distress syndrome (ARDS). Since extracorporeal membrane oxygenation (ECMO) is frequently used in ARDS, but data on CIRCI during this condition are scarce, this study reports the behaviour of adrenal function parameters during oxygenation support with veno-venous (vv)ECMO in coronavirus disease 2019 (COVID-19) ARDS. A total of 11 patients undergoing vvECMO due to COVID-19 ARDS at the Medical University of Vienna, who received no concurrent corticosteroid therapy, were retrospectively included in this study. We analysed the concentrations of cortisol, aldosterone, and angiotensin (Ang) metabolites (Ang I-IV, Ang 1-7, and Ang 1-5) in serum via liquid chromatography/tandem mass spectrometry before, after 1 day, 1 week, and 2 weeks during vvECMO support and conducted correlation analyses between cortisol and parameters of disease severity. Cortisol concentrations appeared to be lowest after initiation of ECMO and progressively increased throughout the study period. Higher concentrations were related to disease severity and correlated markedly with interleukin-6, procalcitonin, pH, base excess, and albumin during the first day of ECMO. Fair correlations during the first day could be observed with calcium, duration of critical illness, and ECMO gas flow. Angiotensin metabolite concentrations were available in a subset of patients and indicated a more homogenous aldosterone response to plasma renin activity after 1 week of ECMO support. Oxygenation support through vvECMO may lead to a partial recovery of adrenal function over time. In homogenous patient collectives, this novel approach might help to further determine the importance of adrenal stress response in ECMO and the influence of oxygenation support on CIRCI.

Faecal cortisol levels in a wild Iberian red deer population are best explained by prior weather conditions

AbstractThe responsiveness of the hypothalamic–pituitary‐adrenocortical axis to stressors is crucial for wild animals to survive and adapt to environmental changes without compromising individual welfare. We analysed the influence of prior weather conditions, seasonality, the influx of ecotourism, and nutrition on stress levels in a wild population of red deer in a Mediterranean hunting reserve in the Pre‐Pyrenees, Spain. We used faecal cortisol metabolites as a proxy for physiological and psychological stress, and faecal triiodothyronine metabolites as an indicator of nutritional stress. Faecal analyses were chosen because it is a non‐invasive technique that does not alter an individual's behaviour, and it is easy to conduct in a wild population, something which presents added challenges due to the lack of a controlled environment. Our results indicate that prior weather conditions, rather than just seasonality, best explained the variations in these hormones within seasons and among years. On the contrary, the results showed that high levels of the cortisol hormone did not necessarily correspond to low levels of the triiodothyronine hormone. This could be a sign of acute nutritional stress in this population. Finally, we did not find an effect of the ecotourism influx on cortisol levels, suggesting that this deer population tolerates the presence of tourists.

How do grazing beef and dairy cattle respond to virtual fences? A review.

Virtual fencing (VF) is a modern fencing technology that requires the animal to wear a device (e.g., a collar) that emits acoustic signals to replace the visual cue of traditional physical fences (PF) and, if necessary, mild electric signals. The use of devices that provide electric signals leads to concerns regarding the welfare of virtually fenced animals. The objective of this review is to give an overview of the current state of VF research into the welfare and learning behavior of cattle. Therefore, a systematic literature search was conducted using two online databases and reference lists of relevant articles. Studies included were peer-reviewed and written in English, used beef or dairy cattle, and tested neck-mounted VF devices. Further inclusion criteria were a combination of audio and electrical signals and a setup as a pasture trial, which implied that animals grazed in groups on grassland for 4h minimum while at least one fence side was virtually fenced. The eligible studies (n = 13) were assigned to one or two of the following categories: animal welfare (n studies = 8) or learning behavior (n studies = 9). As data availability for conducting a meta-analysis was not sufficient, a comparison of the means of welfare indicators (daily weight gain, daily lying time, steps per hour, daily number of lying bouts, and fecal cortisol metabolites [FCM]) for virtually and physically fenced animals was done instead. In an additional qualitative approach, the results from the welfare-related studies were assembled and discussed. For the learning behavior, the number of acoustic and electric signals and their ratio were used in a linear regression model with duration in days as a numeric predictor to assess the learning trends over time. There were no significant differences between VF and PF for most welfare indicators (except FCM with lower values for VF; P = 0.0165). The duration in days did not have a significant effect on the number of acoustic and electric signals. However, a significant effect of trial duration on the ratio of electric-to-acoustic signals (P = 0.0014) could be detected, resulting in a decreasing trend of the ratio over time, which suggests successful learning. Overall, we conclude that the VF research done so far is promising but is not yet sufficient to ensure that the technology could not have impacts on the welfare of certain cattle types. More research is necessary to investigate especially possible long-term effects of VF.

Blend of cinnamaldehyde and diallyl disulfide associated or not to antibiotics on ruminal fermentation, cortisol and blood metabolites of feedlot steers fed no-forage diet

Blend of cinnamaldehyde and diallyl disulfide associated or not to antibiotics on ruminal fermentation, cortisol and blood metabolites of feedlot steers fed no-forage diet