Comparison between blood serum and salivary cortisol concentrations in horses using an adrenocorticotropic hormone challenge

Reasons for performing study: In horses, serum cortisol concentration is considered to provide an indirect measurement of stress. However, it includes both free and bound fractions. The sampling method is also invasive and often stressful. This is not the case for salivary cortisol, which is collected using a more welfare‐friendly method and represents a part of the free cortisol fraction, which is the biologically active form. Objectives: To compare salivary and serum cortisol assays in horses, in a wide range of concentrations, using an adrenocorticotropic hormone (ACTH) stimulation test, in order to validate salivary cortisol for stress assessment in horse. Methods: In 5 horses, blood samples were drawn using an i.v. catheter. Saliva samples were taken using swabs. Cortisol was assayed by radioimmunoassay. All data were treated with a regression method, which pools and analyses data from multiple subjects for linear analysis. Results: Mean ± s.d. cortisol concentrations measured at rest were 188.81 ± 51.46 nmol/l in serum and 1.19 ± 0.54 nmol/l in saliva. They started increasing immediately after ACTH injection and peaks were reached after 96 ± 16.7 min in serum (356.98 ± 55.29 nmol/l) and after 124 ± 8.9 min in saliva (21.79 ± 7.74 nmol/l, P<0.05). Discharge percentages were also different (225% in serum and 2150% in saliva, P<0.05). Correlation between serum and salivary cortisol concentrations showed an adjusted r²= 0.80 (P<0.001). The strong link between serum and salivary cortisol concentrations was also estimated by a regression analysis. Conclusions: The reliability of both RIAs and regression found between serum and salivary cortisol concentrations permits the validation of saliva‐sampling as a noninvasive technique for cortisol level assessment in horses.     

Behavioral and physiological responses to stabling in naive horses

The purpose of this study was to investigate the response of horses to confinement and isolation in a stable (indoor individual housing) for the first time using behavioral indices, heart rate, and salivary cortisol concentration. Six naive 2-year-old Australian Stock Horse fillies were examined at 4-hour intervals over 24 hours in an outdoor group paddock followed by 24 hours in indoor individual housing. Behavioral observations and scores and heart rates were recorded and saliva samples were taken at each interval. During stabling, all horses became agitated and demonstrated increased vocalization and movement. Behavioral scores were significantly higher in the indoor individual housing (P < .001). No significant difference in heart rates between the two environments was detected. Mean salivary cortisol did not increase significantly (2 ng/mL ± 1.4 ng/mL in outdoor group paddock vs 2.5 mL ± 1.2 ng/mL in indoor individual housing). No diurnal rhythm in salivary cortisol was evident in either the outdoor group paddock or indoor individual housing. The results of this study highlight that a combination of behavioral and physiological measures allow better understanding of stress, where one measurement may be misleading. First time stabling of horses elicited marked behavioral responses indicative of stress that were not reflected in increased heart rates or salivary cortisol concentrations. The lack of a diurnal cortisol rhythm and the comparatively high basal cortisol concentrations found in the outdoor group paddock environment may imply that the fillies were already stressed; therefore, stabling did not cause further aberrations detectable by salivary cortisol analysis.     

Circadian variations in salivary chromogranin a concentrations during a 24-hour period in dogs

The purpose of this study was to determine if salivary chromogranin a secretion in dogs exhibits a circadian rhythm. Saliva sampling was performed during three different sessions occurring in three nonconsecutive 24-h periods. Sixteen healthy adult beagle dogs (8 males and 8 females) were moved to a sampling room and housed individually in cages. Saliva samples were obtained every 4 h from 12:00 p.m. to 12:00 p.m. the following day. In the interest of habituation, saliva was obtained hourly from each dog 3 h before the experiment was started. Salivary chromogranin A concentrations were measured using an enzyme-linked immunosorbent assay. No circadian rhythm was detected for salivary chromogranin A secretion, and no differences in salivary chromogranin A concentrations measured every 4 h were demonstrated during the 24-h cycle in dogs.     

Evaluation of the circadian rhythm of anti-Leishmania IgG2 and IgA antibodies in serum and saliva of dogs with clinical leishmaniosis

In this study, the circadian rhythm of IgG2 and IgA specific antibodies in serum and saliva samples of 6 dogs experimentally infected with Leishmania infantum was assessed. Sampling was performed at 8.00, 12.00, 16.00, 20.00, and 00.00 h on two consecutive days. Anti-Leishmania antibody levels in serum were expressed without any correction, whereas in saliva were shown in different ways: without any correction, adjusted by protein concentration and corrected by the salivary flow rate. No significant differences in anti-Leishmania IgG2 antibody levels in serum and saliva samples with or without correction were found. Significant differences were found when anti-Leishmania IgA levels were corrected by the salivary flow rate. In addition, a greater intra-individual variation of antibody levels was observed in saliva than in serum. However, this variation did not modify the serological status of the dogs. Therefore, it could be concluded that there is no circadian rhythm in serum and saliva samples and sampling can be performed at any time of the day.     

Effect of transportation on cortisol concentrations and on the circadian rhythm of cortisol in gilts

A study was conducted to determine whether the circadian rhythm of cortisol in gilts is disrupted or altered by transport. Sixteen ovariectomized gilts with indwelling jugular catheters were individually penned in an enclosed building (location 1). Blood samples were collected at 0700 and 1900 hours for 6 days. On day 7, gilts in groups of 4 were transported 5.6 km to environmentally controlled chambers (25 C) and were individually penned (location 2). On the day of transport, samples were collected at 0700 hours at location 1, immediately before and after transport in a trailer, after unloading at location 2, and at 1900 hours at location 2. For the first 6 days at location 2, blood samples were collected daily at 0700 and 1900 hours. For the 6 days at location 1, circadian rhythm was evidenced by higher cortisol concentrations in the AM hours than in the PM hours. During transport, serum cortisol concentrations increased (P less than 0.01). Highest concentrations developed at 0.5 hour after unloading; concentrations declined thereafter. During the first 6 days at location 2, circadian rhythm was evidenced by higher serum cortisol concentrations in the AM hours than in the PM hours. Therefore, the transportation of gilts 5.6 km to new pens was a transient stress causing a temporary increase in serum cortisol concentrations, but did not cause a disruption in the endogenous rhythm of cortisol.     

Salivary Cortisol Concentration in Exercised Thoroughbred Horses

Both physical activity and stress result in an increase in plasma cortisol level. The measurement of cortisol in plasma requires taking blood samples, which is stressful itself. Therefore, the aim of this study was to evaluate the use of saliva sampling for the determination of cortisol concentrations, indicating the intensity of exercise in horses during race training. Twelve Thoroughbred horses aged 2-3 years were examined during their speed training sessions. The horses galloped on the 1,200-m sand track at a speed of 14.4-15.3 m/s. Three saliva samples and three blood samples were collected from each horse. Both types of samples were taken when the horse was at rest, immediately after returning from the track and 30 minutes after the end of exercise. Blood lactic acid (LA) concentration was determined using the enzymatic cuvette test. The concentrations of cortisol in saliva and plasma samples were measured by enzyme immunoassay methods. Statistically significant correlations were found between salivary cortisol level determined 30 minutes after the end of exercise and blood LA concentration obtained immediately after exercise (P = .003) and between salivary and plasma cortisol levels measured 30 minutes after the end of training session (P = .015). The measurement of cortisol concentration in saliva samples taken from race horses 30 minutes after the end of exercise can be recommended for use in practice under field conditions to estimate the level of relative intensity of exercise in race horses.     

Cortisol response of gilts in tether stalls

Several experiments were conducted to evaluate serum cortisol concentrations and the circadian rhythm of this hormone in gilts tethered in stalls. Control animals were penned individually. In the initial experiment, 18 nongravid gilts were placed in tether stalls after being in either tether stalls or individual pens for 2 wk. No significant differences were found in serum cortisol concentrations. In a second experiment, 16 ovariectomized gilts were placed in tether stalls or individual pens for up to 5 wk. Estrus was induced during wk 3 and 4. During the first day in tether stalls, serum cortisol concentrations increased (P less than .05) and the circadian rhythm of cortisol was disrupted for 4 d. During estrus, the circadian rhythm of cortisol was interrupted for several days in the gilts, regardless of housing. After 4 wk, morning concentrations of cortisol were higher for gilts in tether stalls. The results indicate that: 1) the initial response to tethering varies according to previous penning and handling experience, 2) although the circadian rhythm of cortisol was either altered or disrupted during estrus, such disruptions were not influenced by type of penning and 3) tether stalls may chronically increase cortisol concentrations in gilts.     

Sources of sampling variation in saliva cortisol in dogs

The main advantage of collecting saliva cortisol as opposed to plasma cortisol is that it is non-invasive and therefore it is now widely used in stress measurement studies on farm animals and dogs. Although a plasma cortisol response to handling associated with blood collection generally occurs at 3 min from the commencement of handling, there is no information in the literature on the time course of the response of salivary cortisol concentration to handling. The aims of these experiments were to (1). determine if there is a response to up to 4 min handling that affects cortisol concentration in saliva and (2). determine the main causes of variation in saliva cortisol in dogs over time. In experiment 1, saliva was collected from six Kelpies at 0 min then 2, 3 or 4 min after the commencement of restraint. There was no handling effect found in up to 4 min sampling time. In experiment 2, saliva was collected from six Labrador Retrievers five times in 2 h (14:00-16:00), three days a week for four weeks. Some of the sources of variation in saliva cortisol over time included between dog variation that varied over a period of days and variation between occasions that affected the group of dogs as a whole.     

Circadian and circannual rhythms of cortisol,ACTH, and α-melanocyte-stimulating hormone in healthy horses

Cosinor analysis was used to evaluate whether pituitary and adrenal hormones exhibit circadian rhythmicity in horses. The effect of season and animal age on their respective rhythms was also determined. In addition, the usefulness of evaluating cortisol rhythmicity for the diagnosis of pituitary pars intermedia dysfunction (PPID) was assessed. Serum cortisol concentrations (P < 0.01), but not plasma ACTH or α-melanocyte-stimulating hormone (α-MSH), showed a significant circadian periodicity in horses. An effect of season on hormone concentration was observed with plasma ACTH and α-MSH concentration greater in the fall and cortisol concentration greater in the spring (P < 0.001). Age did not affect cortisol rhythm, but it did blunt the variation in cortisol concentration in horses, similar to what has been previously reported to occur in aged people and dogs. In addition, our results suggest that clinically and diagnostically normal, non–PPID-affected horses commonly have a loss of cortisol diurnal rhythm. Therefore, measurement of circadian rhythm is not an appropriate diagnostic test for PPID.     

Daily rhythms of serum and salivary parameters in goats

Objective   Determine the daily rhythms of urea, bicarbonate, sodium, potassium, chloride, calcium, magnesium and inorganic phosphorus secretion in the serum and saliva of goats and whether there is a relationship between them.
Design   Ten non-gravid, non-lactating does underwent a 30-day preconditioning period (natural 24-h photoperiod, environmental temperature 19–21°C, relative humidity 40–50%, the same feeding regimen, offered as a single meal at 07:00 hours, and water ad libitum), following which saliva and blood samples were collected from each subject every 4 h for 2 days (starting at 08:00 hours) for measurement of urea and electrolytes.
Results   Data analysis was conducted by one-way repeated measures analysis of variance (ANOVA) and by the single cosinor method. ANOVA showed a significant effect of time (0.001 < P < 0.01) on all the studied parameters in saliva and serum on both days. Cosinor analysis identified the periodic parameters and their acrophases (expressed in hours) during the 2 days of monitoring. Serum and salivary acrophases were all diurnal and were observed between 11:40 and 16:24 hours. No circadian rhythmicity was found for bicarbonate, magnesium or potassium in either saliva or serum.
Conclusion   There was a daily rhythm of secretion for the majority of the measured parameters, suggesting the potential role of saliva as an additional, reliable index for physiological, clinical, nutritional and chronophysiological studies.     

Steroid hormone and luteinizing hormone concentrations in the anestrous sow.

This investigation characterized serum concentrations of luteinizing hormone (LH), estradiol-17 beta (E2), progesterone (P4) and cortisol (C) in anestrous sows. Twenty-two sows that had not returned to estrus within 45 days after weaning (anestrous sows), and ten sows that had returned to estrus within seven days following weaning (cyclic sows) were nonsurgically fitted with indwelling jugular vein cannulae. Blood samples were collected at 6 h intervals for seven days and at 15 min intervals for 8 h on the fifth day after cannulation. Serum LH concentrations were determined in all samples, while C, E2 and P4 levels were quantitated in serum collected at 6 h intervals. Serum P4 concentrations in anestrous sows were consistently less than 0.5 ng/mL, and E2 levels ranged from 10 to 19 pg/mL. Concentrations of LH remained less than 1.0 ng/mL in anestrous sows, whereas a preovulatory LH surge was observed in five of ten cyclic sows. There was a circadian rhythm in mean C levels with C peaks occurring at 0600 or 2400 h and nadir levels observed at 1200 and 1800 h. Few differences in C levels were detected between anestrous and cyclic sows. It was evident that anestrous sows did not exhibit cyclic or predictable variations in steroid hormone concentrations. Unfortunately, the results of this study failed to elucidate the endocrine pathogenesis of the anestrous sow.     

Changes in salivary and plasma cortisol levels in Purebred Arabian horses during race training session

Physical activity and stress both cause an increase in cortisol release ratio. The aim of this study was to evaluate the use of saliva samples for the determination of cortisol concentrations indicating the work‐load level in horses during race training. Twelve Purebred Arabian horses aged 3–5 years were studied during the routine training session. After the warm‐up, the horses galloped on the 800 m sand track at a speed of 12.8 m/s. Three saliva samples, and three blood samples were collected from each horse. Both types of samples were taken at rest, immediately after return from the track and after 30 min restitution. The concentrations of blood lactic acid (LA), and cortisol in saliva and plasma samples were measured and analyzed. Blood LA, plasma and salivary cortisol levels increased significantly after exercise (P < 0.05). Salivary cortisol concentration determined 30 min after the exercise correlated significantly with plasma cortisol level obtained immediately after exercise (P < 0.05) as well as measured 30 min after the end of exercise (P < 0.05). The determination of cortisol concentration in saliva samples taken from racehorses 30 min after the end of exercise can be recommended to use in field conditions to estimate the work‐load in racehorses.     

Heart rate and salivary cortisol concentrations in foals at birth

Heart rate (HR), HR variability (HRV) and salivary cortisol concentrations were determined in foals (n = 13) during the perinatal phase and until 5 months of age. In the fetus, HR decreased from 77 ± 3 beats/min at 120 min before birth to 60 ± 1 beats/min at 5 min before birth (P <0.01). Within 30 min of birth, HR increased to 160 ± 9 beats/min (P <0.01). Salivary cortisol concentrations immediately after birth were 11.9 ± 3.6 ng/mL and within 2 h increased to a maximum of 52.5 ± 12.3 ng/mL (P <0.01). In conclusion, increases in HR and salivary cortisol concentrations in foals are not induced during parturition, but occur immediately after birth.     

Influence of Transportation on the Serum Concentrations of the Cardiac Biomarkers Troponin I and Creatine Kinase-myocardial Band (CK-MB) and on Cortisol and Lactate in Horses

The objective of this study was to determine the influence of transportation on the serum concentrations of the cardiac biomarkers troponin I (cTnI) and creatine kinase-myocardial band (CK-MB) and on cortisol and lactate in horses. For this purpose, 10 horses were transported for 300 km. Blood samples were collected 24 hours before transport (T0), just before transport (T1), during transport at 50 km (T2), 100 km (T3), 200 km (T4), and 300 km (T5). An additional blood sample (T6) was collected 24 hours after transport. The median resting basal cTnI values in the horses were at T0, 0.000 ± 0.007 ng/mL and at T1, 0.01 ± 0.007 ng/mL. The median resting basal CK-MB values in the horses were at T0, 0.19 ± 0.05 ng/mL and at T1, 0.16 ± 0.05 ng/mL. Statistical analyses showed no significant differences of cTnI and CK-MB among the measured values (T0–T6). On the other side, the cortisol and lactate concentrations increased significantly (P < .01) at T2, T3, T3, and T4 compared with the resting values at T0. At T6, cortisol and lactate concentrations had returned to pretransport values, with no statistically significant differences compared with pretransport concentrations. In conclusion, the 300-km transportation of the horses did not influence the levels of the cardiac biomarkers, cTnI and CK-MB. The serum concentrations of cortisol and lactate, on the other side, increased significantly. The possible influence of transportation for longer distances or under more stressful conditions (higher temperature or in horses not used for transportation) on cTnI and CK-MB concentrations needs to be further investigated.     

水牛发情周期生殖激素变化规律及唾液结晶的分析

试验对水牛发情周期血清和唾液中雌二醇(E_2)和孕酮(P_4)的浓度变化规律、水牛唾液结晶与卵泡发育变化分别进行了分析研究,为进一步探讨水牛发情规律、指导生产提供依据。采用酶联免疫分析法(ELISA)测定发情母水牛血清和唾液中E_2和P_4的浓度变化,并对血清和唾液的激素变化规律进行相关性分析。结果表明,水牛血清和唾液中的E_2和P_4呈波动性变化。发情前期,唾液中P_4浓度一直维持在6.50～7.10 ng/mL,发情第13天达到11.09 ng/mL,随后快速下降。唾液中E_2浓度在发情第3～5天出现一个峰值178.53 pg/mL,在第14～17天唾液中E_2浓度显著升高,出现第二个峰值179.10 pg/mL。母水牛唾液中E_2和P_4浓度的变化趋势与其在血清中的变化趋势基本一致,均呈显著相关(P<0.05);唾液中E_2与P_4浓度呈极显著相关(P<0.01)。水牛发情当天唾液结晶呈现明显的蕨类作物形状且分维值显著低于其他时间点(P<0.05)。水牛发情周期唾液结晶图形的变化与卵巢卵泡发育基本同步,可作为监测水牛发情及预测排卵的可靠指标之一。     

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.     

Influence of lamb presence on daily rhythm in lactating ewes

Abstract

To determine the influence of lamb presence on the dairy ewes circadian system we monitored simultaneously 16 different parameters (insulin, glucose, total protein, urea, cholesterol, triglycerides, total bilirubine, calcium, phosphorus, magnesium, sodium, potassium, chloride, rectal temperature, respiratory rate and heart rate). Sixteen clinically healthy Sarda breed dairy ewes, divided into two groups, group A with their lambs and group B without their lambs, were used. Data collection was done at 3 hourly intervals over a 24-h period. Our results indicate that lamb presence influences ewes metabolism, inducing modifications of the acrophase and robustness of the rhythm of some physiological variables. Most variables lost the rhythmicity that they show in not pregnant and not lactating ewes. Lamb presence also influenced urea circadian rhythm: the time of urea peak was shifted from 16:00 to 12:24±00:35 when ewes were housed with lambs, and on triglycerides circadian rhythm.     

Changes in Eye Temperature and Stress Assessment in Horses During Show Jumping Competitions

High stress levels in horses during sporting events can influence the animal’s performance in competitions as well as their welfare. This study investigated the use of infrared thermography (IRT) measurement as a rapid, noninvasive, and accurate method for stress assessment in horses during sport competitions and compared IRT with a commonly used physiological method used for stress assessment and salivary cortisol. IRT and salivary cortisol responses were measured 3 hours before the competition, immediately following the competition, and 3 hours after the competition, each day during the 3 days of a show jumping event. Salivary cortisol values ranged from −0.11 ± 4.51 ng/mL in 5-year-old animals to 1.10 ± 2.20 ng/mL in 4-year-old animals, whereas eye temperature values ranged from 35.84 ± 1.16°C in 6-year-old animals to 36.08 ± 1.11°C in 5-year-old animals. The results obtained suggested that IRT measurements constitute an effective method for detecting stress in horses subjected to the acute events of show jumping.     

Evaluation of effects of olfactory and auditory stimulation on separation anxiety by salivary cortisol measurement in dogs

Separation anxiety (SA) is a serious behavioral problem in dogs. In this study, salivary cortisol was studied to determine if the owner''s odor or voice could reduce SA in dogs. Twenty-eight dogs with SA were divided into three groups: group 1 (control), group 2 (with owner''s clothes during the separation period; SP) and group 3 (a recording of the owner''s voice was played during SP). The dog''s saliva was collected after the owner and their dog were in the experimental room for 5 min (PRE). The dog was then separated from the owner for 20 min and saliva collected four times at intervals of 5 min (SP1–4). Finally, the owner was allowed back into the room to calm the dog for 5 min, after which saliva was collected (POST). Evaluation of salivary cortisol concentrations by ELISA revealed that the ratios of SP1 concentration to PRE or POST concentrations were significantly higher in group 1 than in group 2 or 3. Additionally, the concentrations of SP1–PRE and SP1–POST among groups differed significantly. These findings indicate that the owner''s odor or voice may be helpful to managing stress in dogs with SA.     

Factors affecting the circadian rhythm in plasma cortisol concentrations in the horse

In horses, a circadian rhythm in plasma cortisol concentrations has been reported in some but not all studies. When a rhythm occurred, horses were accustomed to a management routine, comprising stabling, feeding and sometimes exercise, which may entrain a circadian pattern. In this work, we monitored plasma cortisol by collecting jugular blood through indwelling cannulae from four groups: 1): 10 untrained, unperturbed mares grazing excess pasture, bled hourly for 26 hr; 2) 4 mares housed in a barn for 48 hr before sampling every 15 min for 20–24 hr; 3) 5 mares placed in an outdoor yard for sampling every 30 min from 0930–2100 hr; and 4) 4 stabled racehorses in training, bled every 30 min from 0730–2000 hr and once the following morning at 0830 hr.Plasma cortisol showed a similarly-timed circadian rhythm (P<0.0001) in all Group 1 horses, with a peak at 0600–0900 hr, and a nadir at 1800–2100 hr. By contrast, cortisol concentrations did not vary with time in either Group 2 or 3. Neither daily mean nor peak cortisol values differed in Group 1 and 2 (i.e. bled for ≥ 20 hr); however nadir values were higher (P<0.05) in Group 2. In Group 4, cortisol declined (P=0.004) during the sampling period but had returned to initial concentrations the next morning. Values did not differ from those for Group 1, except between 1000 and 1300 hr when cortisol in Group 4 was lower (P<0.05).We conclude that a circadian cortisol rhythm exists in horses in the absence of any known cues imposed by humans. However, this rhythm can be obliterated by the minor perturbation of removing the horse from its accustomed environment. By contrast, the rhythm occurs in trained racehorses, suggesting either that they have adapted to their environment thereby allowing an endogenous rhythm to emerge, or that the rhythm is entrained by their daily routine. These observations highlight the difficulties in determining the cortisol status of a horse, since measurements will be affected by time of day, the occurrence of short-term fluctuations, and how accustomed the horse is to its environment.