Validation study of canine serum cortisol measurement with the Immulite 2000 Xpi cortisol immunoassay

We report the results of validation of canine serum cortisol determination with the Immulite 2000 Xpi cortisol immunoassay (Siemens), with characterization of precision (CV), accuracy (spiking-recovery [SR] bias), and observed total error (TEo = bias + 2CV) across the reportable range, specifically at the most common interpretation thresholds for dynamic testing. Imprecision increased at increasing rate with decreasing serum cortisol concentration and bias was low, resulting in increasing TEo with decreasing serum cortisol concentration. Inter-laboratory comparison study allowed for determination of range-based bias (RB) and average bias (AB). At 38.6 and 552 nmol/L (1.4 and 20 μg/dL), between-run CV was 10% and 7.5%, respectively, and TEo ~30% and ~20%, respectively (TEo remained similar regardless of the considered bias: SR, RB, or AB). These analytical performance parameters should be considered in the interpretation of results and for future expert consensus discussions to determine recommendations for allowable total error (TEa). Importantly, the commonly used thresholds for interpretation of results were determined ~40 y ago with different methods of measurements and computation, hence updating is desirable. Quality control material (QCM) had between-run imprecision of 4% for QCM1 and 7% for QCM2; the bias was minimal for both levels. Acceptable QC rules are heavily dependent on the desired TEa for the QCM system (TEa_QCM), itself limited by the desired clinical TEa. At low TEa_QCM (20–33%), almost no rules were acceptable, whereas at high TEa_QCM (50%), almost all rules were acceptable; further investigation is needed to determine which TEa_QCM can be guaranteed by simple QC rules.     

Total observed error,total allowable error,and QC rules for canine serum and urine cortisol achievable with the Immulite 2000 Xpi cortisol immunoassay

Determining a simple quality control (QC) rule for daily performance monitoring depends on the desired total allowable error (TEa) for the measurand. When no consensus TEa exists, the classical approach of QC rule validation cannot be used. Using the results of previous canine serum and urine cortisol validation studies on the Immulite 2000 Xpi, we applied a reverse engineering approach to QC rule determination, arbitrarily imposing sigma = 5, and determining the resulting TEa for the QC material (QCM; TEa_QCM) and the resulting probability of error detection (P_ed) for each QC rule. For the simple QC rule 1_2.5S with P_ed = 0.96 and probability of false rejection (P_fr) = 0.03, the associated TEa_QCM were 20% and 35% for serum and 28% and 24% for urine QCM1 and QCM2. If these levels of TEa_QCM are acceptable for interpretation of patient sample results, then users can internally validate the 1_2.5S QC rule, provided that their QCM CVs and biases are similar to ours. Otherwise, more stringent QC rules can be validated by using a lower sigma to lower the TEa_QCM. With spiked samples (relevant cortisol concentrations in the veterinary patient matrix) at 38.6 and 552 nmol/L of cortisol, TEa_QCM at sigma = 5 were much higher (54% and 40% for serum; 90.3% and 42.8% for urine). Spiked samples generate TEa that is probably too high to be suitable for daily QC monitoring; however, it is crucial to verify spiked sample observed total error (TEo; 26% and 18% for serum, 60% and 30% for urine) < TEa_QCM, and to use spiked sample TEo for patient result interpretation. In the absence of consensus TEa for cortisol in dogs, we suggest the use of a 1_2.5S rule, provided that users accept the associated level of TEa_QCM also as clinical TEa for results interpretation.     

Urinary cortisol‐creatinine ratio in dogs with hypoadrenocorticism

BackgroundBasal serum cortisol (BSC) ≥2 μg/dL (>55 nmol/L) has high sensitivity but low specificity for hypoadrenocorticism (HA).ObjectiveTo determine whether the urinary corticoid:creatinine ratio (UCCR) can be used to differentiate dogs with HA from healthy dogs and those with diseases mimicking HA (DMHA).AnimalsNineteen healthy dogs, 18 dogs with DMHA, and 10 dogs with HA.MethodsRetrospective study. The UCCR was determined on urine samples from healthy dogs, dogs with DMHA, and dogs with HA. The diagnostic performance of the UCCR was assessed based on receiver operating characteristics (ROC) curves, calculating the area under the ROC curve.ResultsThe UCCR was significantly lower in dogs with HA (0.65 × 10⁻⁶; range, 0.33‐1.22 × 10⁻⁶) as compared to healthy dogs (3.38 × 10⁻⁶; range, 1.11‐17.32 × 10⁻⁶) and those with DMHA (10.28 × 10⁻⁶; range, 2.46‐78.65 × 10⁻⁶) (P < .0001). There was no overlap between dogs with HA and dogs with DMHA. In contrast, 1 healthy dog had a UCCR value in the range of dogs with HA. The area under the ROC curve was 0.99. A UCCR cut‐off value of <1.4 yielded 100% sensitivity and 97.3% specificity in diagnosing HA.Conclusions and Clinical ImportanceThe UCCR seems to be a valuable and reliable screening test for HA in dogs. The greatest advantage of this test is the need for only a single urine sample.     

Changes in adrenal cortisol secretion as reflected in the urinary cortisol/creatinine ratio in dogs

In Experiment 1, voided urine samples were collected from 12 adult dogs at 0500, 1400, and 2200 hr for 4 days. Cortisol was measured in unextracted urine by radioimmunoassay, creatinine by spectrophotometry, and the cortisol/creatinine ratio (UCCR) was calculated for each sample. There was considerable variation both within and among dogs in UCCR but there was no consistent time of day fluctuation in UCCR. In Experiment 2, these dogs were randomly assigned to 1 of 4 groups. The groups received each of 4 treatments (saline, dexamethasone, ACTH gel, and aqueous ACTH) at 7 day intervals in a Latin square design. All urine was collected from 0 through 8 hr. Blood samples were collected at 20 minute intervals from 0 through 8 hr. Plasma cortisol exposure was determined by quantifying area under the curve (AUC). UCCR measurement was shown to differentiate basal from elevated, but not lowered, cortisol secretion. A positive linear relationship between UCCR and AUC was seen for all treatments except dexamethasone. These results indicate that changes in cortisol secretion are reflected in changes in UCCR, but measurement of UCCR may lack sensitivity to differentiate basal from reduced states of cortisol secretion. In Experiment 3, urine was collected daily before and during induction therapy with o,p′-DDD from dogs with pituitary-dependent hyperadrenocorticism. Successful suppression of the adrenal glands was accompanied by a progressive decrease in UCCR. There was considerable variation in the rate of adrenal suppression.     

Urinary corticoid to creatinine ratios using IMMULITE 2000 XPi for diagnosis of canine hypercortisolism

The urinary corticoid to creatinine ratio (UCCR) is one of the most commonly used screening tests for canine hypercortisolism (HC). In this study, a reference interval was established for UCCR using IMMULITE 2000 XPi, the latest chemiluminescence enzyme immunoassay. The diagnostic performance of this method for UCCR in canine HC was also evaluated. The median UCCR was 1.06 × 10⁻⁵ (range: 0.28–2.49) for 58 healthy dogs, and an upper reference limit of 1.98 × 10⁻⁵ (90% confidence interval: 1.76–2.15) was determined. The median UCCR in the 12 dogs with HC (7.38 × 10⁻⁵, range 1.86–29.98) was significantly higher than that in the 16 dogs with mimic-HC (1.59 × 10⁻⁵, range 0.47–3.42, P<0.001). The area under the curve for UCCR to differentiate HC dogs from mimic-HC dogs was 0.971, with a sensitivity of 91.7% and specificity of 100% when the cut-off value was set at 3.77 × 10⁻⁵. The UCCR of 16 paired urine samples collected at home and in hospital showed that the UCCR of samples collected in the hospital was significantly higher than that of samples collected at home (mean difference 3.30 × 10⁻⁵, 95% confidence interval: 0.70–5.90, P=0.001). In summary, we established the upper reference limit for UCCR using IMMULITE 2000 XPi in dogs and confirmed that UCCR is a useful diagnostic test for HC in dogs if urine samples are collected at home.     

Evaluation of resting cortisol concentration testing in dogs with chronic gastrointestinal signs

BackgroundResting cortisol concentrations are routinely measured in dogs with chronic gastrointestinal signs to rule out hypoadrenocorticism based on a concentration >2 μg/dL (>55 nmol/L).Hypothesis/ObjectivesTo assess the cross‐sectional prevalence of hypoadrenocorticism in a group of dogs with chronic gastrointestinal signs presented to a referral internal medicine service.AnimalsTwo‐hundred and eighty‐two client‐owned dogs with chronic gastrointestinal signs and with resting cortisol concentration testing performed.MethodsRetrospective review of medical records (final diagnosis, resting cortisol concentration, and adenocorticotropic hormone [ACTH] stimulation test results) of a referral population of dogs between May 2013 and September 2017.ResultsResting cortisol concentration was <2 μg/dL (<55 nmol/L) in 79 patients (28%). Repeated resting cortisol concentration measurements were performed in 28 dogs, and in 8, resting cortisol concentrations remained <2 μg/dL (<55 nmol/L). Post‐ACTH cortisol concentration was <2 μg/dL (<55 nmol/L) in 1 dog, consistent with a diagnosis of hypoadrenocorticism and giving a prevalence estimate of hypoadrenocorticism in this population of dogs of 0.3% (95% confidence interval [95CI], 0.03‐1.5%). In 19 dogs with an initial resting cortisol concentration <2 μg/dL (<55 nmol/L), hypoadrenocorticism was excluded based on a repeat resting cortisol concentration >2 μg/dL (>55 nmol/L). Overall, the most common diagnosis was chronic primary inflammatory enteropathy (176/282, 62.4%), followed by extragastrointestinal neoplasia (17/282, 6%), protein‐losing enteropathy, pancreatitis and megaesophagus (10/282, 3.5% each).Conclusions and Clinical ImportanceAlthough dogs with hypoadrenocorticism can present with chronic gastrointestinal signs, it was the final diagnosis in only 1 of 282 dogs presenting to a referral internal medicine service for signs of chronic enteropathy. Repeated resting cortisol concentration may be considered as a test to try and exclude hypoadrenocorticism.     

The acute effects of a protein‐rich meal on the urinary corticoid:creatinine ratio in healthy dogs

The objective of this prospective crossover study was to investigate the effects of a high‐protein diet on canine urinary corticoid‐to‐creatinine ratio (UCCR). The hypothesis was that meal‐induced hypercortisolism is, as has been shown in humans, a predictable and consistent finding in healthy dogs. Eight clinic‐owned beagles were randomly assigned to one of two groups. The allocation to the groups defined the sequence of a protein‐enriched meal (meal A) or no meal on the first and second days, whereas on the third day all dogs again received an identical meal (meal B) to test reproducibility. Urinary corticoids were measured using a solid‐phase, competitive CLIA on unextracted urine. Contrary to our expectations, consistent incremental responses of the UCCR were not observed (meal A vs. no meal [anova ]: absolute increase, F = 2.546, p = 0.162; relative increase, F = 4.084, p = 0.09; AUC_(UCCR), F = 0.279, p = 0.616). Nevertheless, the robust increases in two dogs above 60% of baseline suggest that the collection of urine prior to feeding likely increases the specificity of the UCCR to discriminate between dogs with and without hypercortisolism.     

Effect of storage time on the urine protein: creatinine ratio in alkaline ovine urine

The urine protein:creatinine (UPC) ratio is considered the reference method to assess proteinuria. Its diagnostic value in ovine medicine needs further elucidation. In population monitoring and/or for research purposes, it is convenient to collect many samples simultaneously and store them for later analysis. However, analyte stability data are required to ensure reliable results. We used 15 of 90 urine samples collected from sheep to assess the effect of storage time on the UPC ratio. After centrifugation, the supernatant of each sample was divided into 6 aliquots. Urine protein and creatinine concentrations were determined immediately in one aliquot using the pyrogallol red and a modified Jaffè method, respectively. The other aliquots were stored at −18°C. Based on the absence of active sediment, alkaline urine pH, and UPC ratio ≥0.2, we included 15 samples in our study. The UPC ratio was determined in the stored aliquots 2, 7, 14, 21, and 60 d after collection. The data were analyzed with univariate ANOVA. No significant difference was observed in the urinary concentrations of protein, creatinine, and the UPC ratio (0.8 ± 0.84 in conventional units and 0.09 ± 0.095 in SI units) among different times (p > 0.05). The UPC ratio remained stable for 2 mo in ovine urine samples stored at −18°C.     

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.     

Urinary glucocorticoid excretion in the diagnosis of hyperadrenocorticism in ferrets

Hyperadrenocorticism in ferrets is usually associated with unaltered plasma concentrations of cortisol and adrenocorticotropic hormone (ACTH), although the urinary corticoid/creatinine ratio (UCCR) is commonly elevated. In this study the urinary glucocorticoid excretion was investigated in healthy ferrets and in ferrets with hyperadrenocorticism under different circumstances. In healthy ferrets and in one ferret with hyperadrenocorticism, approximately 10% of plasma cortisol and its metabolites was excreted in the urine. High-performance liquid chromatography (HPLC) revealed one third of the urinary corticoids to be unconjugated cortisol; the other peaks mainly represented cortisol conjugates and metabolites. In 21 healthy sexually intact ferrets, the UCCR started to increase by the end of March and declined to initial values halfway the breeding season (June). In healthy neutered ferrets there was no significant seasonal influence on the UCCR. In two neutered ferrets with hyperadrenocorticism the UCCR was increased, primarily during the breeding season. In 27 of 31 privately owned ferrets with hyperadrenocorticism, the UCCR was higher than the upper limit of the reference range (2.1 x 10(-6)). In 12 of 14 healthy neutered ferrets dexamethasone administration decreased the UCCR by more than 50%, whereas in only 1 of the 28 hyperadrenocorticoid ferrets did the UCCR decrease by more than 50%. We conclude that the UCCR in ferrets primarily reflects cortisol excretion. In healthy sexually intact ferrets and in ferrets with hyperadrenocorticism the UCCR increases during the breeding season. The increased UCCR in hyperadrenocorticoid ferrets is resistant to suppression by dexamethasone, indicating ACTH-independent cortisol production.     

Interobserver reliability of canine urine specific gravity assessed by analog or digital refractometers

Refractometry is utilized routinely to evaluate canine urine specific gravity (USG) in veterinary clinical settings. We aimed to determine if the magnitude of interobserver reliability when assessing canine USG via refractometry could impact clinical judgment. USG was determined in 38 dogs by 3 registered veterinary technicians (RVTs) using both an optical analog refractometer and a digital refractometer. Summary statistics were reported, interobserver reliability was assessed via intraclass correlation coefficient (ICC) analysis through a 2-way mixed-effects model, and agreement between RVT pairs was compared through Bland–Altman plots. The median analog refractometer USG measurement was 1.018 (range: 1.004–1.040) and for the digital refractometer was 1.0176 (1.0035–1.0357). The analog refractometer average measure ICC was 0.995 (95% CI: 0.992, 0.997; p < 0.001). The digital refractometer average measure ICC was 0.999 (95% CI: 0.999, 1.000; p < 0.001). Strong agreement between all pairs of RVTs was seen via Bland–Altman plots for both analog and digital refractometers, with 95% CIs spanning no more than 0.002 in either the positive or negative direction for all pairings. The interobserver variability in canine USG measurements by RVTs was trivial and did not impact clinical judgment and decision-making.     

Aggression and cortisol levels in three different group housing routines for lactating sows

Background

Lactating sows in Swedish organic piglet production are commonly group-housed with piglets in a multi-suckling pen within 14 days after farrowing. Nursing behaviour may be disturbed when lactating sows are moved to a new environment and mixed with other sows, as they spend more time fighting with other sows and exploring the new surroundings. This can disrupt the inhibitory effect of suckling on ovarian activity and increase the risk of lactational oestrus, making efficient reproductive management difficult. Therefore this study evaluated aggression and levels of the stress hormone cortisol in lactating sows group-housed together with their piglets at one (W1), two (W2) or three (W3) weeks post farrowing.

Results

There was no significant difference (P > 0.05) between the three management routines (W1, W2, W3) regarding number of attacks initiated or received in the mixed group. After mixing, W2 sows had a lower number of shoulder scratches (P < 0.05) than W3 sows. Among the W3 sows, there was a lower (P < 0.01) cortisol concentration in saliva when sows were group housed compared to when they were individually housed. The cortisol response, measured as variation in cortisol concentration in saliva, was also lower (P < 0.05) in group-housed W3 sows compared with W1 sows.For all management routines, sows already living in the new environment (resident sows) initiated more attacks (P < 0.001) and received fewer attacks (P < 0.01) than sows entering the new environment (intruder sows). Overall, multiparous sows initiated more attacks and received fewer attacks than primiparous sows (P <0.001).

Conclusions

Overall, the results suggest that mixing and group housing sows at three weeks post farrowing is less stressful than mixing and group housing sows at one week post farrowing. The results also indicate that parity and whether a sow is a resident or intruder in the group housing environment may have an effect on aggression levels when sows are group-housed.     

Limitations of salivary and blood cortisol determinations in pigs

Blood and saliva samples were taken from groups of pigs maintained in intensive conditions. Multiple samples were taken from two unrestrained pregnant sows fitted with jugular cannulae. Single samples were taken from groups (mixed gilts and entire males; 70–90 kg) which were lightly exercised (7) or restrained (12). The rate of salivary secretion was low and collection of adequate samples took 5 min; in a number of pigs no saliva could be obtained.In order to stimulate salivary secretion, pigs (70–80 kg) were injected with pilocarpine nitrate (25 mg, subcutaneous) which produced a copious flow of saliva persisting for at least 15 min.Resting sows had higher mean levels of cortisol in plasma, ultrafiltrate and saliva than the other groups, which did not differ from each other. Within the pilocarpine group, males had a higher ultrafiltrate level of cortisol than females. In most instances salivary cortisol was significantly greater than ultrafiltrate cortisol.Ultrafiltrate and plasma cortisol were highly correlated (r=0.883) but this correlation was low in the presence of pilocarpine (r=0.260). Salivary cortisol was poorly correlated with either plasma (r=0.167) or ultrafiltrate cortisol (r=0.278) and the correlation with plasma was even lower following the administration of pilocarpine (r=0.086).It was concluded that salivary estimates of cortisol in the pig were not usefully correlated with levels of ultrafiltrate (free) cortisol.     

Monitoring the circadian rhythm of serum and salivary cortisol concentrations in the horse

Daily fluctuations of cortisol concentration in the blood or saliva have been repeatedly reported. However, several contradictions in the existing literature appear on this subject. The present study was performed to definitively establish options for testing adrenocortical function. To the best of our knowledge, this is the first study to evaluate parallel circadian rhythms in salivary and serum cortisol concentrations during a 24-h period. Twenty horses were examined under the same conditions. Blood and saliva samples were taken every 2 h for 24 h to determine the daily changes in cortisol concentrations of saliva and serum at rest and to determine the relationship between salivary and serum cortisol levels. Cosinor analysis of group mean data confirmed a significant circadian component for both serum and salivary cortisol concentrations (P < 0.001 in both cases). The serum cortisol circadian rhythm had an acrophase at 10:50 AM (95% CI, 10:00 AM–11:40 AM), a MESOR of 22.67 ng/mL, and an amplitude of 11.93 ng/mL. The salivary cortisol circadian rhythm had an acrophase at 10:00 AM (95% CI, 9:00 AM–11:00 AM), a MESOR of 0.52 ng/mL, and an amplitude of 0.12 ng/mL. We found a significant but weak association between salivary and serum cortisol concentrations; the Pearson correlation coefficient was 0.32 (P < 0.001). The use of salivary cortisol level as an indicator of hypothalamic-pituitary-adrenal axis activity may be warranted. However, the salivary cortisol levels are more likely to be correlated with free plasma cortisol than with the total plasma cortisol concentration.     

Study on cortisol,cortisone and prednisolone presence in urine of Chianina cattle breed

The Chianina, one of the oldest and most important cattle breeds of Italy, is now reared all over the world. The Chianina has been known and appreciated since ancient times because, from a nutritional point of view, its meat has no proper rivals. To date, studies have been performed to evaluate the genetic profile of the breed, but knowledge about the chemical profile is generally lacking. Due to the increased interest from farmers regarding breeding of the Chianina, this study proposes a preliminary evaluation of main endogenous urinary corticosteroids (cortisol and cortisone) and most commonly used synthetic one (dexamethasone). Moreover, after recent findings regarding the presence of endogenous prednisolone in the urine of more popular breeds, particular attention was given to analysis of the presence of prednisolone and prednisone, as well. For this aim, the urine samples of 12 young cows and 30 young bulls was collected at the farms and analysed using a fit‐for‐purpose LC‐MS/MS method. The preliminary results of this study show that prednisolone was found only in Chianina females (3 out of 12). Cortisol and cortisone were found at concentrations that showed a high inter‐individual variability, and that were higher in female urine compared to that of males.     

Speckle tracking echocardiography in mature Irish Wolfhound dogs: technical feasibility,measurement error and reference intervals

Background

Two-dimensional strain measurements obtained by speckle tracking echocardiography (STE) have been reported in both humans and dogs. Incorporation of this technique into canine clinical practice requires the availability of measurements from clinically normal dogs, ideally of the same breed, taken under normal clinical conditions.The aims of this prospective study were to assess if it is possible to obtain STE data during a routine echocardiographic examination in Irish Wolfhound dogs and that these data will provide reference values and an estimation of measurement error.

Methods

Fifty- four healthy mature Irish Wolfhounds were used. These were scanned under normal clinical conditions to obtain in one session both standard echocardiographic parameters and STE data. Measurement error was determined separately in 5 healthy mature Irish Wolfhounds.

Results

Eight dogs were rejected by the software algorithm for reasons of image quality, resulting in a total of 46 dogs (85.2%) being included in the statistical analysis. In 46 dogs it was possible to obtain STE data from three scanning planes, as well as to measure the rotation of the left ventricle at two levels and thus calculate the torsion of the heart. The mean peak radial strain at the cardiac apex (RS-apex) was 45.1 ± 10.4% (n = 44), and the mean peak radial strain at the base (RS-base) was 36.9 ± 14.7% (n = 46). The mean peak circumferential strain at the apex (CS-apex) was -24.8 ± 6.2% (n = 44), and the mean peak circumferential strain at the heart base (CS-base) was -15.9 ± 3.2% (n = 44). The mean peak longitudinal strain (LS) was -16.2 ± 3.0% (n = 46). The calculated mean peak torsion of the heart was 11.6 ± 5.1 degrees (n = 45).The measurement error was 24.8%, 26.4%, 11.5%, 6.7%, 9.0% and 10 degrees, for RS-apex, RS-base, CS-apex, CS-base, LS and torsion, respectively.

Conclusions

It is concluded that this technique can be included in a normal echocardiographic examination in large breed dogs under clinical conditions. The usefulness of the reference values reported here, given their wide normal range, will ultimately be determined by the values that are obtained from a large numbers of diseased dogs.     

Serum cortisol in mules intended for agricultural work

Cortisol is a hormone secreted by the adrenal glands that is stimulated by physiological and pathological factors and has been studied widely in equids, but not in mules. The objectives of this study were to obtain a reference value for serum cortisol in mules destined for agricultural activities and to identify age- and gender-related differences in the value. The concentration was obtained in blood samples using a commercial sandwich ELISA specific for cortisol. The concentration was 96.3 ± 40.6 ng/ml and similar between age groups and genders.     

Hydrocortisone Concentrations in Post-Race Urine from Horses

As hydrocortisone is an endogenous substance, it is first necessary to establish its normal concentrations so as to be able to control its use in racing animals. This study was designed to establish the hydrocortisone concentrations in post-race urine samples of horses racing in Brazil and also to evaluate the results in relation to the international threshold set for this drug. Urine samples were analysed by HPLC-UV. The results were evaluated according to the concentration range as well as sex and time of sample collection (afternoon or evening races). The results showed a high degree of variation in the concentrations of hydrocortisone in the urine (93±69 ng/ml). The maximum concentration observed was 646 ng/ml, although only a few horses (around 1%) showed levels within the range 500–650 ng/ml, 91% being in the range 0–150 ng/ml. The data suggested a normal distribution curve. Statistical analysis showed no significant influence of sex or time of sample collection.     

Reference intervals for electrophoretograms obtained by capillary electrophoresis of dialyzed urine from healthy dogs

Electrophoresis of urine to evaluate protein fractions in dogs with proteinuria to differentiate glomerular from tubular damage has increased in recent years; however, capillary electrophoresis (CE) of urine has not been reported in a study of > 40 healthy animals, to our knowledge. We aimed to establish reference intervals (RIs) for the urine protein fractions obtained by CE of urine from healthy dogs. We obtained urine samples from 123 clinically healthy dogs of both sexes between December 2016 and April 2019; urine was frozen until CE was performed. The electrophoretic patterns obtained were divided into 5 protein fractions, and RIs were established in percentages and absolute values using nonparametric methods. RIs were obtained for the fractions (F) as follows: 5.5 to 56.2% for F1, 3.2 to 16.5% for F2, 3.5 to 16.2% for F3, 17.8 to 69.8% for F4, and 5.1 to 23.9% for F5. These RIs obtained by CE might be useful clinically as a basis for comparison with pathologic samples. Age was a statistically significant factor for F2 (p = 0.01) and F3 (p = 0.02), and sex was a statistically significant factor for F1 (p = 0.03).     

Time lag between peak concentrations of plasma and salivary cortisol following a stressful procedure in dairy cattle

Background

Measurement of salivary cortisol has been used extensively as a non-invasive alternative to blood sampling to assess adrenal activity in ruminants. However, there is evidence suggesting a considerable delay in the transfer of cortisol from plasma into saliva. Previous studies in cattle have used long sampling intervals making it difficult to characterise the relationship between plasma and salivary cortisol (PLCort and SACort, respectively) concentrations at different time points and determine whether or not such a time lag exist in large ruminants. Therefore, the objective of this study was to characterise the relationship between plasma and salivary cortisol and determine if there is a significant time lag between reaching peak cortisol concentrations in plasma and saliva across a 4.25 h time-period, using short sampling intervals of 10–15 min, following social separation in dairy cattle.Five cows were separated from their calves at 4 days after calving, and six calves were separated from a group of four peers at 8 weeks of age. Following separation, the animals were moved to an unfamiliar surrounding where they could not see their calves or pen mates. The animals were catheterised with indwelling jugular catheters 1 day before sampling. Blood and saliva samples were obtained simultaneously before and after separation.

Results

In response to the stressors, PLCort and SACort increased reaching peak concentrations 10 and 20 min after separation, respectively. This suggested a 10 min time lag between peak cortisol concentrations in plasma and saliva, which was further confirmed with a time-series analysis. Considering the 10 min time lag, SACort was strongly correlated with PLCort (P < 0.0001).

Conclusions

Salivary cortisol correlates well with plasma cortisol and is a good indicator of the time-dependent variations in cortisol concentrations in plasma following acute stress. However, there is a time lag to reach peak cortisol concentrations in saliva compared to those in plasma, which should be considered when saliva samples are used as the only measure of hypothalamic-pituitary-adrenal axis response to stress in cattle.