Sham Calves Research Articles

479 Pain control for the castration of newborn bull calves by elastration

Abstract All methods of castration are known to cause pain regardless of the method, or age of the animal. Our objective was to explore providing pain control for bull calves castrated by elastration prior to 1 wk of age. Singleton, bull calves (n = 85) born at the Ontario Beef Research Centre were enrolled and randomly assigned to 1 of 5 treatment groups at birth: a single dose of oral meloxicam at castration (1ORAL), a dose of oral meloxicam at castration and a second oral dose 1 wk later (2ORAL), a dose of injectable meloxicam at castration (1INJECT), not castrated (SHAM), or castrated without pain control (PAINFUL). Calves were treated within 48 h of birth and followed until weaning. All calves were observed 2 h/d for 13 different pain-associated behaviors (e.g., tail flicks, kicking, licking the scrotum) on the day of castration (d 0), as well as d 1, 2, 7, 8, and 9. To record general activity, from d -1 to d 14 each calf wore an Onset HOBO Pendant G Data Logger attached to one of their hind legs. Calves were weighed weekly until they were turned-out to pasture and thereafter weighed bi-weekly until weaning. Five behaviors were statistically significant between treatment groups. PAINFUL calves had roughly three times the rate of licking (P > 0.05) compared with calves in 1ORAL, 2ORAL, and 1INJECT groups, and four times the rate of licking compared with SHAM calves. Additionally, PAINFUL calves had two times the rate of tail flicks (P < 0.05) compared with meloxicam-treated calves, and five times the rate of tail flicks compared with SHAM calves. A treatment effect (P < 0.05) was observed for standing time, with PAINFUL calves spending the least amount of time standing on average in a 24-h period. No statistical time differences were seen between the remaining treatment groups. No treatment or treatment*time interaction effects were observed for lying time. A tendency was seen for treatment effect on lying time (P = 0.0581), with PAINFUL calves spending the most time lying on average and having a significance difference compared with all other treatment groups (LSM > 0.05). No treatment effect was seen for average daily gain up to wk 8, and for final weaning weights. Weight at time of castration was significant (P < 0.05); however, calves were not blocked by BW when assigned to treatment. In conclusion, meloxicam-treated calves expressed decreased rates of pain-associated behaviors compared with PAINFUL calves, and BW did not differ at time of weaning. Our results provide evidence that calves castrated by elastration before 1 wk of age and received meloxicam, experience less pain.

Assessing dairy calf response to long-distance transportation using conditioned place aversion

Surplus neonatal dairy calves are routinely transported long distances from the dairy farm of birth to calf raisers. Most of the research assessing the effect of transportation on young calves has focused on physiology and health, without considering calf affective states. The aim of this study was to assess the affective response of young male dairy calves to transportation using conditioned place aversion (CPA) testing. Three bouts of transportation exposed dairy calves (n = 95) to transportation for 6 (n = 31), 12 (n = 32), or 16 h (n = 30). During transportation, the trailer interior was made visually distinct using blue horizontal (n = 2 groups) or red vertical (n = 1 group) stripes. Distinct color and pattern combinations were used to create a visual association with transportation. A subset of calves (6 h = 14; 12 h = 14; 16 h = 14) were selected for CPA testing if they were ≥7 and <28 d of age at the time of testing after transport. A group of sham calves that were not transported (n = 13) and were ≥7 and <28 d of age at the time of testing also were enrolled. Wall coverings with the same color and pattern of those in the trailer were fixed in individual sham calf stalls for 6 h on the morning of transportation. Before transportation, calves were placed in a 3-compartment test apparatus with neutral walls and were able to move about freely for 15 min to habituate them to the test apparatus. Calves were CPA tested on d 2 after transportation. During the posttest, calves were placed in the test apparatus with 3 compartments including a neutral center pen (center), blue horizontal stripe compartment (side), and red vertical stripe compartment (side) for 30 min; calves were able to move throughout the apparatus freely. Time spent in each compartment was recorded using continuous observation and analyzed as a percentage of total time in the apparatus. Calves spent less time in the compartment associated with transportation compared with the compartment with no prior association (22% vs. 42% ± 3% of time in the transportation-association and no-association compartments, respectively); however, this behavior was not affected by transportation treatment. Results from this study suggest that calves may perceive transportation to be aversive regardless of duration but may also be attracted to exploring a novel stimulus.

Buffering lidocaine heightens aversion to cornual nerve injections in dairy calves

Best practice is to numb the horn buds before disbudding dairy calves, which can be achieved by injecting lidocaine, a local anesthetic, around the cornual nerve. In humans, the acute pain that occurs upon injection of lidocaine can be reduced by neutralizing the pH of the solution with an alkalizing agent, such as sodium bicarbonate. We evaluated whether buffering lidocaine would reduce calves' behavioral and physiological responses to cornual nerve blocks on the left and right sides of the head. Female Holstein calves were assigned to 1 of 3 treatments: cornual nerve blocks with 5.5 mL of unbuffered lidocaine (n = 9), buffered lidocaine (n = 9), or a sham procedure with no needle inserted (n = 9). Calves that received either type of lidocaine struggled more during the injection than sham calves. However, contrary to our hypothesis, struggling was most marked in calves that received buffered lidocaine. Similarly, calves administered unbuffered or buffered lidocaine had elevated heart rates for 1 or 3 min after the first injection, respectively, compared with at the end of the 5-min observation period. Calves in the buffered treatment had lower eye temperatures in the first half of the observation period compared with the second half, consistent with responses cattle show to other aversive procedures, but no changes over time were observed in the other 2 treatments. We detected no treatment differences in heart rate variability measures. These results suggest that cornual nerve blocks are aversive, at least in the short term, and that buffering the lidocaine worsens the calf's response to this procedure.

Social approach and place aversion in relation to conspecific pain in dairy calves.

Despite scientific interest in animal empathy, and growing public concern for farm animal welfare, the empathic abilities of farm animals remain under researched. In this study, we investigated empathic responses of young Holstein dairy calves to conspecifics recovering from hot-iron disbudding, a painful procedure common on dairy farms. A combination of social approach and place conditioning was used. First, ‘observer’ calves witnessed two ‘demonstrator’ calves recover from either a painful procedure (hot-iron disbudding and sedation) or a sham procedure (sedation alone) in distinct pens. Observer calves spent more time in proximity and paid more attention to calves recovering from the painful procedure compared to sham calves (proximity: 59.6 ± 4.3%; attention: 54.3 ± 1.5%). Observers were then tested for conditioned place aversion (in the absence of demonstrators) at 48h, 72h and 96h after the second demonstration; observers tended to avoid the pen associated with conspecific pain during the second of the three tests, spending 34.8 ± 9.6% of their time in this pen. No strong evidence of pain empathy was found, but our tentative results encourage further research on empathy in animals.

Conditioned place preference reveals ongoing pain in calves 3 weeks after disbudding

Hot-iron disbudding, a routine procedure that prevents horn bud growth through cauterization, is painful for calves. The resulting burns remain sensitive to touch for weeks, but it is unknown whether calves experience ongoing, non-evoked pain. We evaluated conditioned place preference for analgesia in 44 calves disbudded or sham-disbudded 6 hours (Day 0) or 20 days (Day 20) before testing (n = 11/treatment). Calves were conditioned to associate the effects of a lidocaine cornual nerve block with the location and pattern of a visual stimulus, and a control injection of saline with the contrasting stimulus. On Day 0, disbudded calves tended to prefer the lidocaine-paired stimulus over the saline-paired one, suggesting that they found analgesia rewarding. On Day 20, sham calves avoided the lidocaine-paired stimulus, consistent with humans’ experience of this drug being painful. Disbudded calves on Day 20 did not show this aversion, suggesting that they traded off the short-term pain of the lidocaine with the longer-term analgesia provided. Day 0 sham calves did not avoid the lidocaine-paired stimulus, likely because they received less than half the dose of Day 20 calves during conditioning. Thus, higher doses of lidocaine are aversive to uninjured animals, but disbudded calves are willing to engage in this cost. We conclude that calves experience ongoing pain 3 weeks after disbudding, raising additional welfare concerns about this procedure.

Short communication: Assessment of disbudding pain in dairy calves using nonlinear measures of heart rate variability

The purpose of this study was to evaluate whether pain-related stress caused by disbudding could be detected using nonlinear measures of heart rate variability (HRV). Twenty-five female Holstein calves (4-7 wk of age) were randomly assigned to 1 of 3 treatments: (1) sham disbud (SHAM; n = 9), (2) disbud with lidocaine-meloxicam pain mitigation (MED; n = 8), or (3) disbud without pain mitigation (NoMED; n = 8). Heart rate variability (sample entropy, percentage determinism, percentage recurrence, or mean length of diagonal lines in a recurrence plot) was recorded on d -1, 0, 1, 3, and 5 relative to the experimental procedure, with disbudding taking place on d 0. The short-term detrended fluctuation analysis scaling exponent was greater in MED calves than in SHAM calves, indicating a greater stress response to the disbudding procedure regardless of pain mitigation. These results indicate that calves in the MED group may have experienced pain-related stress as a result of the disbudding procedure. The remaining nonlinear HRV measures did not differ by treatment. Future research on this topic should address additional potential confounding factors, such as the effect of pain-mitigating drugs on autonomic function or the influence of the autonomic nervous system on wound healing, that may prohibit HRV measurement as an indicator of disbudding pain severity.

Effect of Lignocaine and a Topical Vapocoolant Spray on Pain Response during Surgical Castration of Beef Calves.

Simple SummaryEnsuring a good state of welfare in farm animals is important for ethical reasons and sustainability of livestock industries. Addressing pain in farm animals during and following surgical husbandry procedures is a significant component of animal welfare. Until recently, practical constraints associated with delivery of traditional forms of analgesia have prevented widespread uptake by producers, especially in large, extensively managed animal production systems. In recent years, novel anaesthetic and analgesic products have been developed to facilitate practical delivery of post-operative pain relief to livestock. However, the issue of intra-operative pain during husbandry procedures remains unaddressed. Therefore, this study investigated the efficacy of pre-operative injected lignocaine and peri-operative topical vapocoolant spray, administered as most practical for incorporation into routine procedures, for pain relief during castration of calves. Results from this study provide no evidence that lignocaine or vapocoolant spray reduced pain during castration of calves. Pulling of the spermatic cords appeared to be the most painful component of the castration procedure. This finding may assist in clarifying what methods for relieving procedural pain associated with castration of calves merit future research.This study assessed the efficacy of pre-operative injected lignocaine and peri-operative topical vapocoolant spray, administered as most practical for incorporation into routine calf castration procedures. Calves were randomly allocated to: (1) sham castration (SHAM); (2) surgical castration (CAST); (3) surgical castration with lignocaine (LIG); and (4) surgical castration with vapocoolant spray (VAPO). Calf behavioural responses were scored at different stages of the sham castration or castration procedure. Maximum ocular temperatures were measured at three time-points relative to restraint and treatment. There were significant effects of treatment (p < 0.001) and stage of procedure (p < 0.001) on calf behavioural response. SHAM calves were more likely to display less severe responses compared to all other calves and LIG calves were more likely to display less severe responses compared to VAPO calves. Calves were more likely to display more severe responses to extrusion of the first spermatic cord compared to all other stages of castration, and to extrusion of the second spermatic cord compared to severing of the second spermatic cord. There was a significant effect of time (p < 0.001) on ocular temperature, with ocular temperature being greater following sham castration or castration. In this study, there was no evidence of pain reduction during castration of calves by either lignocaine or vapocoolant spray.

The effect of disbudding age on healing and pain sensitivity in dairy calves

Hot-iron disbudding, a husbandry procedure performed in dairy calves in which horn bud growth is prevented through tissue cauterization, produces severe burns. Little is known about how long pain persists following disbudding or factors, such as age at the time of disbudding, that may influence animal welfare outcomes. Our objectives were (1) to evaluate whether disbudding age mediates healing, pain sensitivity, and weight gain in calves disbudded near birth (3 d) compared with at a later age (35 d); and (2) to examine how long wounds are sensitive during healing. Forty-eight calves were disbudded or sham-disbudded at 3 or 35 d of age (n = 12/treatment). We assessed pain sensitivity with a pressure algometer on d 3 and 7 after disbudding and then weekly thereafter until the wound had re-epithelialized. Sensitivity was measured at the lateral and caudal edges of each wound, as well as on the rump to test for systemic changes in sensory function. We evaluated healing by scoring the presence of 7 tissue types in the wound bed: attached necrotic tissue, detached necrotic tissue, burns outside the necrotic ring, exudate, granulation, crust, and epithelium. Tissue scores, surface temperature, and girth circumference as an estimate of weight gain were measured twice weekly until the wound had re-epithelialized. Disbudded tissue was more sensitive than nondisbudded tissue for at least 3 wk, after which time the sham calves were removed from the study to be disbudded. Wounds took 62 ± 10 d (mean ± SD) to re-epithelialize and were more painful than new epithelium throughout this time, regardless of whether the procedure occurred at 3 or 35 d of age. The surface temperature of disbudding wounds did not differ from that of non-disbudded tissue nor did disbudding affect weight gain at either age. Age at the time of disbudding did not affect latency to re-epithelialize or wound sensitivity. However, rump sensitivity was greater in calves disbudded at 3 versus 35 d of age on d 28, 42, and 56 after disbudding, in agreement with other studies showing increased pain sensitivity when painful procedures are performed at an early age. In conclusion, wounds remain painful throughout healing, raising animal welfare concerns about hot-iron disbudding. Performing the procedure near birth does not improve welfare outcomes; rather, some evidence suggests it may produce a generalized long-term increase in pain sensitivity.

Measurement of dairy calf behavior prior to onset of clinical disease and in response to disbudding using automated calf feeders and accelerometers

We determined if feeding and lying behavior, recorded by automatic calf feeding systems (ACFS) and accelerometers, could be used to detect changes in behavior before onset of neonatal calf diarrhea (NCD) or in response to disbudding pain in dairy calves. At 4 d of age, 112 calves had accelerometers attached to their hind leg and were housed in pens with ACFS. Calves were examined daily for signs of illness or injury. Of the 112 calves monitored, 18 were diagnosed with NCD; activities of calves with NCD were then compared with those of 18 healthy controls (calves that had no symptoms of NCD, other illnesses, or injury). Feeding (milk consumption and the number of rewarded and unrewarded visits to the feeder) and lying behavior during the 5 d leading up to calves displaying clinical signs of NCD were analyzed. Calves with NCD performed fewer unrewarded visits and consumed less milk than healthy calves during the 2- and 4-d periods before diagnosis with NCD, respectively. Calves with NCD tended to perform fewer lying bouts than healthy calves over the 5-d period before diagnosis with NCD. At 3 wk of age, a subset of 51 healthy calves were allocated to 1 of 5 treatment groups: (1) sham handling (SHAM, n = 10), (2) cautery disbudding (DB, n = 11), (3) administration of local anesthetic (LA) and DB (LA+DB, n = 11), 4) administration of a nonsteroidal anti-inflammatory drug (NSAID) and DB (NSAID+DB, n = 9), and (5) administration of LA, NSAID and DB (LA+NSAID+DB, n = 10). Feeding and lying behavior were recorded continuously for 24 h pre- and postdisbudding. We found no effect of treatment on the number of rewarded or unrewarded visits to the feeder and milk volume consumed 24 h before administration of treatments. During the 24-h postdisbudding period, SHAM calves performed more unrewarded visits than DB, LA+DB, and NSAID+DB calves, but the number of unrewarded visits did not differ between SHAM and LA+NSAID+DB calves. During the first hour of the posttreatment period we noted a difference in lying times among treatments, with DB and NSAID+DB calves spending less time lying than SHAM calves and lying times being similar between SHAM, LA+DB, and LA+NSAID+DB calves. The ACFS and accelerometers have the potential to automatically gather valuable information regarding health status and pain in calves. Therefore, it may be advantageous to combine both of these measures (ACFS and accelerometers) when evaluating NCD on farm or pain in calves in future research.

Effects of topical anaesthetic and buccal meloxicam on average daily gain, behaviour and inflammation of unweaned beef calves following surgical castration

Although the pain caused by castration of calves is a significant animal welfare issue for the beef industry, analgesia is not always used for this procedure, largely because of practical limitations associated with injectable forms of pain relief. Novel analgesic formulations have now been developed for livestock to allow topical and buccal administration, offering practical options to improve cattle welfare if shown to be effective. To assess the effects of topical anaesthetic (TA) and buccal meloxicam (BM) on average daily gain (ADG), behaviour and inflammation following surgical castration of beef calves, a total of 50 unweaned bull calves were randomly allocated to: (1) sham castration (SHAM, n=10); (2) surgical castration (C, n=10); (3) surgical castration with pre-operative buccal meloxicam (CBM, n=10); (4) surgical castration with post-operative topical anaesthetic (CTA, n=10); and (5) surgical castration with pre-operative buccal meloxicam and post-operative topical anaesthetic (CBMTA, n=10). Calves were recorded on video for 5 h following treatment and the frequency and duration of specific behaviours displayed by each animal was later observed for 5 min every hour (total of 25 min). Average daily gain was calculated 1, 2 and 6 days following treatment. Scrotal diameter measurements and photographs of wounds were collected from all castrated calves 1, 2 and 6 days following treatment to evaluate inflammation and wound healing. Infrared photographs were used to identify maximum scrotal temperature. Digital photographs were used to visually score wounds on a numerical rating scale of 1 to 5, with signs of inflammation increasing and signs of healing decreasing with progressive scores. Sham castration calves displayed significantly less, and C calves displayed significantly more foot stamps than all other calves (P=0.005). Observations on the duration of time that calves displayed a hypometric ‘stiff gait’ locomotion, indicated that SHAM calves tended to spend no time, C calves tended to spend the greatest time and all other calves tended to spend an intermediate time displaying this behaviour (P=0.06). Maximum scrotal temperatures were lower in CBM and CBMTA calves than C and CTA calves 2 days following treatment (P=0.004). There was no significant effect of treatment on ADG (P=0.7), scrotal diameter (P=0.09) or wound morphology score (P=0.5). These results suggest that TA and BM, alone or in combination, reduced pain and BM reduced inflammation following surgical castration of calves.

Welfare outcomes for 3- and 6-month-old beef calves in a tropical environment castrated surgically or by applying rubber rings

Castration of cattle using rubber rings is becoming increasingly popular due to the perceived ease of the procedure and greater operator safety when compared with surgical castration. Few comparative studies have investigated the effects of different castration methods and calf age on welfare outcomes, particularly in a tropical environment. Thirty Belmont Red (a tropically adapted breed), 3-month-old (liveweight 71–119kg) and 30, 6-month-old (liveweight 141–189kg) calves were assigned to a two age×three castration (surgical, ring and sham) treatment factorial study (Surg3, Surg6, Ring3, Ring6, Sham3 and Sham6, n=10 for each treatment group). Welfare outcomes were assessed post-castration using: behaviour for 2 weeks; blood parameters (cortisol and haptoglobin concentrations) to 4 weeks; wound healing to 5 weeks; and liveweights to 6 weeks. More Surg calves struggled during castration compared with Sham and Ring (P<0.05, 90±7% vs. 20±9% and 24±10%) and performed more struggles (1.9±0.2, 1.1±0.3 and 1.1±0.3 for Surg, Sham and Ring, respectively), suggesting that surgical castration caused most pain during performance of the procedure. A significant (P<0.05) time×castration method×age interaction for plasma cortisol revealed that concentrations decreased most rapidly in Sham; the Ring6 calves failed to show reduced cortisol concentrations at 2h post-castration, unlike other treatment groups. By 7h post-castration, all treatment groups had similar concentrations. A significant (P<0.01) interaction between time and castration method showed that haptoglobin concentrations increased slightly to 0.89 and 0.84mg/mL for Surg and Ring, respectively over the first 3 days post-castration. Concentrations for Surg then decreased to levels similar to Sham by day 21 and, although concentrations for Ring decreased on day 7 to 0.76mg/mL, they increased significantly on day 14 to 0.97mg/mL before reducing to concentrations similar to the other groups (0.66mg/mL) by day 21. Significantly (P<0.05) more of the wounds of the 3-month compared with the 6-month calves scored as ‘healed’ at day 7 (74% vs. 39%), while more (P=0.062) of the Surg than Ring scored as ‘healed’ at day 21 (60% vs. 29%). At day 14 there were significantly (P<0.05) fewer healed wounds in Ring6 compared with other treatment groups (13% vs. 40–60%). Liveweight gain was significantly (P<0.05) greater in 3-month (0.53kg/day) than in 6-month calves (0.44kg/day) and in Sham calves (P<0.001, 0.54kg/day), than in Ring (0.44kg/day) and Surg (0.48kg/day) calves. Overall, welfare outcomes were slightly better for Surg than Ring calves due to reduced inflammation and faster wound healing, with little difference between age groups.

Platelet activation, aggregation, and life span in calves implanted with axial flow ventricular assist devices

Background. A variety of rotary blood pumps are under development worldwide to serve as chronic ventricular assist devices (VADs). Historically VADs have been associated with thrombotic and thromboembolic complications, yet the ability to evaluate the thrombotic process in preclinical device testing has been limited. Methods. We have developed and applied flow cytometric assays for activated platelets, platelet microaggregates, and platelet life span and consumption to calves implanted with an axial flow VAD and calves undergoing a sham surgical procedure. Results. Surgical sham calves had significant increases in circulating activated platelets ( p < 0.05) that resolved within 17 days, and no increases in circulating platelet microaggregates. Calves with uneventful VAD implant periods had early transient elevations in platelet microaggregates and prolonged elevations in activated platelets that did not recover to preoperative values during the study. Daily platelet consumption in VAD implanted calves was increased by 20% ± 3%. Calves with thrombotic deposition within the VAD and elevated thromboembolism observed at autopsy experienced increases in circulating activated platelets and microaggregates at the end of the implant period when VAD flow decreased. Conclusions. This study demonstrates the ability of flow cytometry-based platelet assays to differentiate VAD implant operations from VAD support, and suggests differences that exist between uneventful VAD support and support with complications. These techniques should have value in evaluating other cardiovascular devices undergoing preclinical testing and provide insight into the temporal impact of these devices on the hemostatic system.

Effects of prenatal stress on the fetal calf

Twelve pregnant Brahman cows were randomly assigned to one of two treatment groups: 1) transported in a stock trailer for 24.2 km, unloaded at a second farm and penned for 1 hr, and then returned to the original farm (TRANS, n = 6); or 2) walked through the handling facilities (SHAM, n = 6). Treatments were repeated at 60, 80, 100, 120, and 140 d of gestation. Calves were delivered by cesarean section on d 266 of gestation. The male:female ratio was 4:2 and 5:1 for the TRANS and SHAM treatment groups, respectively. Before calf removal and severance of the umbilical blood flow, a blood sample was collected from the calf to determine plasma concentrations of adrenocorticotropin (ACTH) and cortisol. The calf was then sedated and exsanguinated, after which pituitary and adrenal glands were collected. The adrenals were immediately weighed, and a cross-section from the left adrenal was stored in 4% paraformaldehyde until being embedded in paraffin. Eight sections from each adrenal were sliced (5 μm), fixed, and then stained with Harris' hematoxylin and eosin. Areas of the cortex and medulla were calculated with a computerized digitizing unit and tracing of the viewed section. The TRANS calves had heavier body weights (BW) (28.7 vs. 23.9 ± 1.8 kg; P < 0.07), pituitary glands (12.63 vs. 8.24 ± 1.10 g/kg BW; P < 0.008), and heart weights (5.58 vs. 5.17 ± 0.58 g/kg BW; P < 0.05) than did the SHAM calves. Plasma concentrations of ACTH and cortisol did not differ between SHAM and TRANS calves (57 vs. 82 ± 14 pg/ml and 7.0 vs. 6.7 ± 0.9 ng/ml, respectively; P > 0.2). Adrenal gland weight and medulla-to-cortex ratio did not differ between SHAM and TRANS calves (0.61 and 0.73 ± 0.03 g and 0.97 and 0.99 ± 0.12 g, respectively; P > 0.2). These results suggest that the altered response to stress in prenatally stressed calves is not associated with morphological changes in the adrenal gland but may be due to effects of prenatal stress on the fetal pituitary.

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.