Changes In Plasma Cortisol Research Articles

Maternal corticotropin-releasing hormone is increased with impending preterm birth.

The objective of this study was to test the hypothesis that maternal CRH concentrations are elevated in women experiencing threatened preterm labor who subsequently give birth within 24 h compared to those in women who do not. We also characterized the changes in maternal plasma cortisol, ACTH, corticosteroid binding capacity (CBC), and CRH concentrations in 28 healthy pregnant women between 20-38 weeks gestation. Overall, maternal plasma CRH concentrations were significantly greater (P < 0.05) in those women giving birth within 24 h (1343.3 +/- 143.9 pg/mL; n = 81) compared to those in women who did not (714.5 +/- 64.8 pg/mL; n = 144) or those in normal subjects. This difference was present between 28-36 weeks, but not 24-28 weeks gestation. The ratio of maternal cortisol to CBC was also significantly greater (P < 0.05; 0.65 +/- 0.04; n = 82) in women giving birth within 24 h than in those who did not (0.55 +/- 0.02; n = 136). This difference was significant at all gestational ages studied. Elevated CRH concentrations and bioavailability of free cortisol may both be implicated in the pathogenesis of preterm labor in some women. Further prospective clinical trials are warranted to determine the positive and negative predictive values of maternal CRH concentrations and/or the ratio of cortisol/CBC for identifying women with threatened preterm labor destined to give birth within 24 h.

11β-HSD and 17β-HSD as biological markers of depression: Sex differences and correlation with symptom severity

There is growing interest in the hydroxysteroid dehydrogenases (HSDs) as local modulators of hormone action. We have studied urinary steroid profiles from nine men and nine women with Major Depression to determine whether 11β-HSD and 17β-HSD activity are altered. Urinary steroid profiles were determined by high resolution gas chromatography. The ratio of 11-oxo over 11β-hydroxy metabolites of cortisol was used as the index of 11β-HSD activity and the ratio of dehydroepiandrosterone (DHA) over androstenediol-17β was used as the index of 17β-HSD activity. Symptom severity was assessed using the Hamilton Depression Rating Scale (HDRS). None of the measures of cortisol production, 9 AM plasma cortisol, 24 hour urinary free cortisol or 24 hour total urinary cortisol metabolites with HDRS in either men or women. 11β-HSD activity was altered and correlated with symptom severity in depressed women (r=0.688, p<0.05) but not men (r=0.132). In both depressed men and women, 17β-HSD activity was altered and correlated with HDRS scores (r=0.796, p<0.05 and r=0.688, p<0.05 respectively). However, although the ratio was changed in the same direction in depressed men and women, the correlation with symptom severity was positive in women but negative in men. We conclude that markers of subtle change such as these may prove more useful and informative than gross changes in plasma cortisol in depression.

Comparison of methods for the reduction of acute painproduced by rubber ring castration or tail docking of week-old lambs

Behavioural and plasma cortisol changes were recorded for groups of eight Suffolk x Greyface lambs subjected to castration or tail docking using rubber rings with and without local anaesthetic treatment. Immediately after application of the rubber ring, local anaesthetic (2 x 0.2 ml 2% lignocaine) was administered either by needle and syringe or by high-pressure needleless injection into each side of the neck of the scrotum or tail at the site of the ring, or by high pressure needleless injection into the testes before ring application. In other groups, the innervation to the scrotum or tail was disabled by crushing with a powered bloodless castrator just proximal to the ring. Measurements were recorded in groups of control (handled) lambs, with and without local anaesthetic treatment. Application of local anaesthetic by high pressure needleless injection had little effect on either plasma cortisol values or behaviour of control lambs. For castration, application of the bloodless castrator and/or local anaesthetic at the ring site reduced the peak plasma cortisol concentration by 50% (P < or = 0.01), the incidence of active behavioural responses by 80 and 64% (P < or = 0.01) respectively, and the time spent in abnormal postures by 68 and 59% (P < or = 0.01) respectively. Both methods were effective in reducing pain for tail docking [active behavioural responses reduced by more than 80% (P < or = 0.01) and abnormal postures by 56% (P < or = 0.01)], although local anaesthetic was more effective, reducing the peak cortisol by 60% (P < or = 0.01) [crushing by 44% (P > 0.05)]. Injection of local anaesthetic into the testes was less effective than injection into the neck of the scrotum at the site of the ring [reduction in abnormal lying postures (P < or = 0.05), 45 vs 71%, respectively]. The rapid action, effectiveness, and ease of application of these experimental methods may provide the basis for commercially viable methods for reducing the acute pain produced by rubber ring castration and tail docking of lambs.

Behavioral, endocrine, immune, and performance measures for pigs exposed to acute stress.

Weanling pigs (n = 132) were used to investigate the effects of three common stressors (and a control) and differing social status on behavior, immunity, plasma cortisol, blood chemical, and performance measures. Eleven blocks of 12 pigs each were evaluated. Each pen contained three pigs of dominant (DOM), intermediate (INT), or submissive (SUB) social status. Two weeks later, random pens of pigs experienced either a control treatment (CON) or they were stressed for 4 h by shipping (SHIP), heat-stressed (HEAT) with overhead heat lamps in their home pens, or cold-stressed (COLD) by direct application of water and an air current. Treatments did not influence body weights; however, percentage weight loss during SHIP was greater than for other treatments. Body weights were heavier for DOM pigs than for INT and SUB pigs. Social status had large effects on plasma cortisol, globulin, acute-phase proteins, body weight, and weight changes. Only acute shipping stress resulted in weight loss. Many immune and blood measures were not changed among acutely stressed pigs; however, the relationship between social status and mitogen-induced lymphocyte proliferation and natural killer cell cytotoxicity was disrupted during acute stress. Pig behavior was significantly changed by each stress treatment in a unique manner. During acute stress, behavioral changes seem to be the most consistent and reliable indicators.

Depressive response to physostigmine challenge in borderline personality disorder patients

The purpose of this study was to examine the relationship between mood and hormonal responses to cholinergic challenge with physostigmine in order to assess cholinergic system responsiveness in borderline personality disorder (BPD) patients, other non-BPD personality disorder patients, and normal controls. Thirty-four personality disorder patients, 10 of whom met criteria for BPD and 24 of whom met criteria for other, non-borderline, personality disorders, and 11 normal controls participated in a double blind, placebo controlled physostigmine challenge paradigm. The Profile of Mood States depression subscale (POMS-D) self report measure was obtained at baseline and following the physostigmine or placebo infusions. A repeated measures ANOVA of POMS-D scores in placebo and drug conditions indicated a significantly greater depressive response in the total cohort of personality disorder patients than in the normal comparison group (p < 0.05). However, the depressive response to physostigmine was significantly greater in BPD patients, but not other personality disorder patients, compared to normal controls (p < 0.05). There was a correlation between the peak placebo-corrected depressive response to physostigmine and a group of BPD traits related to affective instability but not a group of BPD traits related to impulsivity. There was no correlation in any group between mood response to physostigmine and changes in plasma cortisol, prolactin, or growth hormone, or to nausea or other side effects following physostigmine infusion. These data suggest that there is an association between BPD and acute depressive responses to physostigmine challenge, and that the cholinergic system may be involved in the regulation of affect in Axis II disorders.

Stress in transported sheep

Sheep are widely transported around the world, with low (0.018%) mortality rates for slaughter lambs within the UK but high (2.2%) for lambs shipped from Australia to the Middle East. Transport of animals is a multifactorial stressor, road and driving conditions, noise, vibration, atmospheric conditions, ventilation, intransit space allowance, journey length and water and food deprivation all being potential individual stressors. Changes in heart rate, plasma cortisol and prolactin suggest that sheep are stressed at the beginning of journeys but soon adapt to the conditions provided they have sufficient room to lie down, i.e. τ; 0.25 m2 per lamb or about 140 kg/m2. Plasma creatine kinase activities suggest that the incidence of serious injury and exhaustion are rare in transported lambs. Provided environmental conditions are not excessively hot, sheep cope well with a lack of water on journeys as long as 24 h. However, they do show signs of hunger with increased time spent feeding immediately after journeys longer than 8 h and increases in both free fatty acids and βs-hydroxybutyrate as journeys progress. Providing short rest periods with food and water within a long journey may not be advisable because sheep feed rather than drink or rest and the lamb’s fluid balance may be upset, so lowering sheep welfare. However, a longer (12 h) lairage may be beneficial.

Plasma leptin levels do not change in patients with Cushing's disease shortly after correction of hypercortisolism.

In the present study, we characterized the changes in plasma leptin levels in patients with pituitary Cushing's disease and in age- and sex-matched controls. Plasma levels of ACTH, cortisol, and leptin were measured before and after iv administration of ovine CRH in controls once and in patients twice (while they had active hypercortisolism and 10 days after successful surgery). Cushing's patients had elevated body mass indexes (34 +/- 1.9 vs. 22.9 +/- 0.8) and plasma leptin levels (35.6 +/- 3.4 vs. 9.2 +/- 1.9 ng/mL) compared to controls, which remained unchanged 10 days after successful transsphenoidal surgery and directly proportional to the body mass index. Plasma leptin levels were not affected by CRH infusion in either the controls or the patients despite clear-cut elevations in plasma ACTH and cortisol. These findings suggest that although acute changes in plasma cortisol do not affect plasma leptin, chronic hypercortisolism results in elevated leptin levels, probably by causing visceral obesity.

Carbohydrate supplementation affects blood granulocyte and monocyte trafficking but not function after 2.5 h or running

This randomized, double-blind, placebo-controlled study was designed to determine the influence of carbohydrate supplementation on the granulocyte and monocyte response to 2.5 h of high-intensity running [76.7 +/- 0.4% of maximal oxygen consumption (VO2max)]. Thirty experienced marathon runners (VO2max 53.4 +/- 1.0 mL.kg-1.min-1, age 41.5 +/- 1.4 y) were randomly assigned to carbohydrate-supplement (n = 17) and placebo (n = 13) groups. Subjects rested for 10-15 min before a blood sample was taken at 0715, and then ingested 0.75 L carbohydrate beverage or placebo. At 0730 subjects began running at 75-80% of VO2max for 2.5 h, and drank 0.25 L carbohydrate or placebo fluid every 15 min. Immediately after the 2.5-h run (1000), another blood sample was taken, followed by 1.5-h, 3-h, and 6-h recovery samples. Carbohydrate supplementation had a significant effect compared with placebo on the pattern of change in plasma glucose and cortisol, and the blood concentration of neutrophils (F[14, 112] = 5.13, P = 0.001) and monocytes (F[14, 112] = 4.78, P = 0.001), but not on blood granulocyte and monocyte phagocytosis or oxidative burst activity after 2.5 h of intensive running.

Coincidental changes in behavior and plasma cortisol in unrestrained pigs after intracerebroventricular injection of tumor necrosis factor-alpha.

The coincidental behavioral and physiological responses to inflammatory stimuli administered either peripherally or centrally were evaluated. In the first study, twenty castrated male pigs were injected ip with 0, 0.5, 5, or 50 microg/kg BW lipopolysaccharide (LPS). Body temperature was monitored telemetrically, and serial blood samples were collected via an indwelling jugular catheter for determination of plasma cortisol and tumor necrosis factor-alpha (TNF-alpha) concentrations. Sickness behaviors were measured during 10-min tests at 0, 2, 4, 8, 12, and 24 h post injection. The 5 and 50 microg/kg doses of LPS increased plasma concentrations of cortisol and TNF-alpha, while inducing anorexia, hypersomnia, and fever. In contrast, although 0.5 microg/kg LPS induced acute anorexia, hypersomnia, and fever, it did not increase plasma TNF-alpha; and the cortisol response was small and transient, suggesting the behavioral system in pigs is more responsive to LPS than the hypothalamic-pituitary-adrenal (HPA) axis. Because LPS-induced behavior and activation of the HPA axis involve proinflammatory cytokines in the brain, in a second study, unrestrained pigs with jugular catheters were injected intracerebroventricularly (I.C.V.) with recombinant porcine TNF-alpha. Vehicle or TNF-alpha (0, 5, or 50 ng/kg) was injected I.C.V., and plasma cortisol and behavior were determined as before. Pigs injected I.C.V. with 50 ng/kg TNF-alpha showed anorexia, hypersomnia, and an abrupt increase in plasma cortisol concentration. Whereas 5 ng/kg TNF-alpha I.C.V. also induced marked sickness behavior, it failed to stimulate the HPA axis, as indicated by plasma cortisol levels. That there was a distinct difference in the magnitude of behavioral and endocrine responses to LPS and TNF-alpha suggests that different systems that are responsive to inflammatory stimuli exhibit different sensitivities.

Coincidental Changes in Behavior and Plasma Cortisol in Unrestrained Pigs after Intracerebroventricular Injection of Tumor Necrosis Factor-

Coincidental Changes in Behavior and Plasma Cortisol in Unrestrained Pigs after Intracerebroventricular Injection of Tumor Necrosis Factor-

Reduction in pain suffered by lambs at castration

The acute pain produced by bloodless castrators was studied by comparing the behavioural and plasma cortisol changes in groups (n=8) of 3-week-old Dorset cross lambs after castration with a 22 cm (9") Burdizzo, a new power assisted castrator and by a combined method using a Burdizzo and elastrator ring. The time spent in abnormal postures (52-58 min) and the peak cortisol response (110-120 mmol l(-1)) were similar for the three methods, although the powered castrator produced a more sustained response. The Burdizzo method halved the incidence of active behaviours compared with the powered castrator and combined methods (16 versus 30, 32 counts). Intratesticular local anaesthetic administered 2 min before the Burdizzo castrator and combined method, or intramuscular injection of the non-steroidal anti-inflammatory drug diclofenac, 20 min before the application of a Burdizzo, significantly reduced the peak plasma cortisol response to 80 nmol l(-1). Diclofenac also significantly reduced the time spent trembling or in abnormal postures.

Intravenous vs. oral rehydration: effects on subsequent exercise-heat stress.

This study compared the influence of intravenous vs. oral rehydration after exercise-induced dehydration during a subsequent 90-min exercise bout. It was hypothesized that cardiovascular, thermoregulatory, and hormonal variables would be the same between intravenous and oral rehydration because of similar restoration of plasma volume (PV) and osmolality (Osmo). Eight non-heat-acclimated men received three experimental treatments (counterbalanced design) immediately after exercise-induced dehydration (33 degrees C) to -4% body weight loss. Treatments were intravenous 0.45% NaCl (iv; 25 ml/kg), no fluid (NF), and oral saline (Oral; 25 ml/kg). After rehydration and rest (2 h total), subjects walked at 50% maximal O2 consumption for up to 90 min at 36 degrees C. The following observations were made: 1) heart rate was higher (P < 0.05) in Oral vs. iv at minutes 45, 60, and 75 of exercise; 2) rectal temperature, sweat rate, percent change in PV, and change in plasma Osmo were similar between iv and Oral; 3) change in plasma norepinephrine decreased less (P < 0.05) in Oral compared with iv at minute 45; 4) changes in plasma adrenocorticotropic hormone and cortisol were similar between iv and Oral after exercise was initiated; and 5) exercise time was similar between iv (77.4 +/- 5.4 min) and Oral (84.2 +/- 2.3 min). These data suggest that after exercise-induced dehydration, iv and Oral were equally effective as rehydration treatments. Thermoregulation, change in adrenocorticotropic hormone, and change in cortisol were not different between iv and Oral after exercise began; this is likely due to similar percent change in PV and change in Osmo.

Plasma catecholamine and lymphocyte beta 2-adrenergic receptor alterations in elderly Alzheimer caregivers under stress.

The purpose of this study was to determine the effects of chronic stress on beta-adrenergic physiology in elderly spousal caregivers to Alzheimer patients. Thirty-seven elderly spousal caregivers and matched noncaregiver controls (mean age 73 years, SD = 6) were studied. Life stress categorization (presence of marked threat) covering the previous 6 months was determined using a semistructured interview based on the Psychiatric Epidemiological Research Inventory and the Life Events and Difficulties Schedule. beta 2-adrenergic receptor sensitivity (isoproterenol-stimulated cyclic AMP accumulation) and density were determined in lymphocytes. Caregivers with high life stress had higher plasma norepinephrine levels (p < .04) but no change in plasma cortisol. For beta-receptor sensitivity, 30% of the variance was accounted for by high life stress rating, increased age, being male, and lower norepinephrine (p = .018); 17% of the variance in beta-receptor density was accounted for by plasma norepinephrine (p = .03). The findings demonstrate that chronic high stress may be associated with changes in adrenergic physiology and may provide a mechanism through which chronic stress alters cellular immunity.

Effects of prenatal stress on suckling calves.

Pregnant Brahman cows (n = 42), bred to either Brahman or Tuli bulls, were randomly assigned to one of three treatments: 1) transported in a stock trailer for 24.2 km, unloaded at a second farm and penned for 1 h, and then returned to the original farm (TRANS); 2) i.v. injection of ACTH, 1 IU/kg BW (ACTH); or 3) walked through the handling facilities (SHAM). Treatments were initiated on d 60 and repeated at 80, 100, 120, and 140 d of gestation. The calves from these cows were subjected to tests to measure their capacity to react to stress. In Test 1, Tuli-sired calves were restrained at 10 and 150 d of age for 3.5 h. In Test 2, Brahman-sired calves were restrained for 3.5 h and given an injection of ACTH (.125 IU ACTH/kg of BW). In Test 3, Test-2 calves were restrained at 180 d of age and hot-iron branded. In Test 4, Test-1 calves were restrained at 180 d of age and given an injection of cortisol (6.7 ng/kg BW) to estimate cortisol clearance rate. During all tests, calves were restrained for 3.5 h, and heart rates were recorded and blood samples were taken at -15, 0, 15, 30, 45, 60, 90, 120, and 180 min. The 10- and 150-d-old TRANS calves maintained greater plasma cortisol in Test 1 (restraint) than the ACTH and SHAM calves (P < .01). The ACTH challenge (Test 2) increased plasma cortisol and ACTH, but cow treatment did not alter the response (P > .4). In response to branding (Test 3), the TRANS, ACTH, and SHAM calves' overall mean plasma cortisol was not affected by treatment (52, 51, and 43 +/- 3 ng/mL, respectively; P > .1), nor was the calves' overall heart rate (91, 94, and 86 +/- 3 beats/min, respectively; P > . 1). In Test 4, TRANS calves cleared plasma of cortisol at a slower rate than did the SHAM calves (P < .01), but not the ACTH calves (261, 374, and 473 +/- 50 mL/min, respectively; P > .1). The TRANS calves had an overall greater heart rate than did the ACTH or the SHAM calves (91, 79, and 77 +/- 2 beats/min, respectively; P < .001). Exposing cows to repeated transportation stress during gestation altered their calf's physiological response to stress, and these alterations could have a profound influence on the calfs ability to adapt to stress, thereby influencing its welfare. Further research should examine the growth, immune function, and reproductive function of prenatally stressed calves to determine whether these changes in plasma cortisol are beneficial or deleterious.

Activation of the fetal hypothalamic-pituitary-adrenal axis with prolonged and graded hypoxemia.

The purpose of this study was to determine whether activation of the hypothalamic-pituitary-adrenal (HPA) axis and uteroplacental prostaglandin (PG) response occur before metabolic deterioration with a progressive decrease in fetal oxygenation. Twenty-one chronically catheterized fetal sheep were studied between 126 and 135 days' gestation during a 24-hour normoxic control period and subsequently during 4 experimental days of either prolonged and graded hypoxemia, induced by progressively lowering the maternal inspired oxygen concentration (induced hypoxia, n = 12), or continued study on room air (control, n = 6; spontaneous hypoxia, n = 3). Fetal arterial blood was sampled daily for blood gases and pH, immunoreactive ACTH, cortisol, and PGE2. Placental cotyledons were obtained at the end of the experiment for measurement of prostaglandin-H synthase (PGHS) enzymatic activity. For all hypoxia group measurements, progressive reduction in fetal oxygenation resulted in little change in either plasma ACTH or cortisol until arterial O2 saturation was close to 30% with metabolic acidosis onsetting. This was in keeping with activation of the HPA axis at this time, because ACTH and cortisol values showed a strong linear correlation (r = 0.77, P < .01). Fetal plasma PGE2 concentrations and cotyledonary PGHS enzymatic activity, although somewhat higher in the hypoxia group animals, were not changed significantly. In response to prolonged and graded hypoxemia in the ovine fetus, activation of the HPA axis occurs only when the degree of hypoxemia is pronounced and close to that associated with metabolic deterioration, which may limit the time for any uteroplacental response and the ability to initiate labor.

Endocrine response to lactate infusion during pentobarbitone anaesthesia

Lactic acid was infused iv in 6 Welsh ponies during pentobarbitone anaesthesia to investigate whether lactate triggers the pituitary-adrenal response to anaesthesia. Ponies were premedicated with acepromazine and anaesthesia was induced with pentobarbitone iv and maintained with pentobarbitone/oxygen for 2 h. Immediately after induction, 3% L(+) lactic acid infusion was started and adjusted to maintain plasma lactate concentration between 2 and 2.5 mmol/l. Cardiorespiratory function, temperature. PCV, plasma glucose, lactate, βendorphin, ACTH, cortisol and catecholamine concentrations were measured before, during and after anaesthesia. Hypothermia, reduced PCV, slight hypotension (minimum value 84 ± 6 mmHg 20 min after induction of anaesthesia), hyperoxia and marked bradypnoea developed during anaesthesia. No acidaemia occurred. Plasma glucose concentration increased at the end of anaesthesia. There were no changes in plasma ACTH, cortisol and catecholamine concentrations, but plasma & endorphin increased after induction until the end of anaesthesia. There was a correlation between plasma lactate and β-endorphin concentrations (P<0.001, r=0.63), which may suggest that lactate stimulates βendorphin release. Beta-endorphin was apparently secreted independently from ACTH and appears to be a sensitive marker of a stress response.

Differential regulation of proopiomelanocortin messenger ribonucleic acid in the pars distalis and pars intermedia of the pituitary gland after prolonged hypoxemia in fetal sheep.

POMC messenger RNA (mRNA) is synthesized and translated to form ACTH in the pars distalis and ACTH-related peptides in the pars intermedia of the pituitary in fetuses of species such as sheep. In the fetal sheep, maturation of hypothalamo-pituitary-adrenal (HPA) function occurs during late pregnancy accompanied by increased levels of POMC mRNA in the pars distalis, and contributes to the stimulus for birth. Activation of fetal HPA function also occurs with short term hypoxemia. However, the effects of more prolonged fetal hypoxemia, which may predispose to premature parturition, on POMC mRNA levels in the pars distalis and pars intermedia are not known. We studied the effects of sustained hypoxemia (48 h) in the absence of acidosis on fetal HPA responses at two times in late gestation, days 126-130 and days 134-136 (term = approximately 145 days). Hypoxemia was induced by lowering the fraction of oxygen in the maternal inspired gas mixture. Fetal arterial oxygen tension fell by 6-8 mm Hg, without a change in fetal arterial pH or carbon dioxide tension. Pituitary POMC mRNA was localized and quantified by in situ hybridization. At both gestational ages, hypoxemia caused a transient elevation in fetal plasma ACTH, with a maximum response at +2 h, and a sustained elevation in circulating cortisol. Cortisol responses were greater in the older fetuses. Changes in plasma cortisol were not associated with alterations in the plasma corticosteroid-binding capacity or in levels of hepatic corticosteroid-binding globulin mRNA. Pituitary POMC mRNA showed a regional distribution in the pars distalis, and mean levels increased with gestational age. After 48 h of hypoxemia, POMC mRNA levels had increased in the pars distalis. The regional distribution was unaffected by the hypoxemic insult. In contrast, POMC mRNA levels in the pars intermedia decreased after hypoxemia, and the reduction was greater in the older fetuses. We conclude that in response to prolonged hypoxemia, there is differential regulation of POMC mRNA levels in the pars distalis and pars intermedia of the pituitary in fetal sheep. The magnitude of these responses also changes with gestational age. The increase in POMC mRNA levels in the pars distalis at 48 h of hypoxemia, despite a rise in plasma cortisol and without a change in corticosteroid-binding globulin, suggests altered regulation of HPA function at this time.

P-15-25 - Changes of plasma prolactin, ACTH, cortisol & clinical symptoms after ECT in schizophrenia

P-15-25 - Changes of plasma prolactin, ACTH, cortisol & clinical symptoms after ECT in schizophrenia

Changes in plasma cortisol, plasma b-endorphin, heart rate, haematocrit and plasma protein concentration in horses during restraint and use of a naso-gastric tube

Changes in plasma cortisol, plasma b-endorphin, heart rate, haematocrit and plasma protein concentration in horses during restraint and use of a naso-gastric tube

Assessment of acute and chronic pain after different methods of castration of calves

Four groups of Ayrshire calves ( n = 8 each) were castrated at 1 week of age by either surgery, crushing by a Burdizzo, rubber ring or a combination of the Burdizzo and rubber ring methods. Responses of castrated calves were compared with a control group ( n = 8) of handled calves. Plasma cortisol was measured in blood samples taken during the first 3 h after castration. Observations of behaviour were made for 3 h immediately after castration, and for 3 h each afternoon for the first 4 days and then on every third day to 48 days after treatment. An assessment of the castration site (lesion score) was made after each observation period and the calves were weighed every twelfth day. Acute changes in behaviour and plasma cortisol were consistent with those reported previously, namely that increases in active behaviours, abnormal postures and plasma cortisol occurred in the first 3 h after castration. The behavioural response to both surgical and Burdizzo castration was characterised by immobile postures. The combined Burdizzo and rubber ring method produced a significant reduction in the activity (count per 3 h period) of two behaviours (foot stamping/kicking (11.9) and easing quarters (13.3)) compared with the use of a rubber ring alone (27 and 23.7, respectively). During the 48 days after rubber ring treatments there was an increase in the calves' concern with the site of castration, indicated by a significantly ( P < 0.01) increased incidence of licking which was associated with increased abnormal standing, with slow movements of the tail, alternate lifting of the hindlegs and head turning as the integrity of the skin broke down. If these indices are valid, they demonstrate that considerable acute and chronic pain is associated with rubber ring methods of castration of calves and that the chronic pain can last for at least 42 days.