TY - JOUR
AU - Schwartzkopf-Genswein, K. S.
AB - ABSTRACT Three experiments were conducted to evaluate the effects of band and knife castration on behavioral and physiological indicators of chronic pain in beef calves at 3 different ages (36 calves/age group): 1 wk of age (Exp. 1, 4 ± 1.1 d of age, 43 ± 1.1 kg BW), 2 mo of age (Exp. 2, 63 ± 2.3 d of age, 92 ± 1.7 kg BW), and 4 mo of age (Exp. 3, 125 ± 4.6 d of age, 160 ± 3.4 kg BW). In each experiment calves were randomly assigned to either sham (CT), band (BA) or knife (KN) castration. Experiments 1, 2, and 3 ended when the testicles of banded calves had sloughed off (68, 49, and 42 d, respectively). Animal BW and rectal temperature were recorded weekly over the experimental period. Salivary cortisol, substance P, haptoglobin, scrotal area temperature using infrared thermography, visual evaluation of swelling (5-point scale), and gait stride length were collected on d −1 and immediately before castration and weekly thereafter until the end of the study. Hair samples were collected 1 d prior to and 28 d after castration and at the end of the study for cortisol concentration. Standing and lying behaviors were recorded over a 28-d period immediately after castration. No differences (P > 0.10) were observed in salivary cortisol, substance P, haptoglobin, or hair cortisol among castration methods for any of the 3 ages. No changes in behavior were observed in calves castrated at 1 wk or 2 mo of age. In 4 mo-old-calves, BA spent less time lying (P < 0.01) than CT and KN calves. Also, the average duration of lying time for BA calves was greater (P < 0.05) than for CT calves. Both, 1-wk- and 2-mo-old calves had inflammation in the scrotal area lasting 7 d after KN castration, whereas inflammation was observed for up to 14 d in 4-mo-old calves. Swelling in BA calves lasted for 21 to 28 d in the 2 younger groups of calves, whereas in 4-mo-old calves swelling was observed until d 35 postcastration. Knife- and band-castrated calves did not exhibit indicators of chronic pain or distress when the procedures were performed in calves younger than 2 mo of age. Therefore, pain mitigation should be used when castrating to improve animal welfare, especially when castrations are performed in calves older than 2 mo of age independent of the method of castration. INTRODUCTION The castration of beef calves is a common practice in North America (National Agricultural Statistics Service, 2015) and usually occurs between 1 d and 1 yr of age (Coetzee et al., 2010). The United States and Canada lack legislation related to optimal age, method, or use of pain control agents at the time of castration (Webster et al., 2013). The Canadian Code of Practice for the Care and Handling of Beef Cattle (National Farm Animal Care Council, 2013) requires that calves be castrated as young as practically possible by competent personnel using clean, well-maintained instruments and accepted techniques. A recent survey of cow-calf operations in western Canada (Moggy et al., 2017) reported that 96% of bulls are castrated before 3 mo of age, whereas in the United States only 59.2% of calves are castrated before sale (USDA, 2008). In addition, Moggy et al. (2017) indicated that only 4% of producers used pain control at these ages, indicating that castration in calves 3 mo of age and younger is typically conducted without the use of pain control (Dockweiler et al., 2013). Knife castration and band castration are the most common methods used (National Agricultural Statistics Service, 2015). In calves younger than 1 wk and older than 3 mo o of age, band castration is the preferred method (99.8% and 60.1%, respectively), whereas in 1-wk- to 3-mo-old calves, knife castration is preferred (60.4%; Moggy et al., 2017). Regardless of method, there is a plethora of studies indicating that all methods cause significant pain during and after the procedure (Molony et al., 1995; Robertson et al., 1994; González et al., 2010; Warnock et al., 2012). Knife castration is suggested to be more painful at the time of the procedure, whereas band castration produces more chronic pain (Molony et al., 1995; Thüer et al., 2007; González et al., 2010). To date, there are few studies that evaluate the chronic or long-term effects of castration associated with method and age. Hence, the aim of this study was to evaluate the effect of band and knife castration on chronic indicators of pain in 1-wk- and 2- and 4-mo-old calves. We hypothesized that younger calves would present fewer indicators of chronic pain after band castration. MATERIALS AND METHODS This paper is part of a larger study evaluating the optimal age and method of castration assessing acute pain (Meléndez et al., 2017) as well as chronic pain, which is described in the present paper. All procedures described within this study were approved by the Animal Care and Use Committee of the Lethbridge Research Centre (ACC1410) and the University of Calgary Animal Care Committee (AC14–0159) in accordance with the guidelines established by the Canadian Council on Animal Care (2009). Animals, Housing, and Management A total of 108 Angus and Angus crossbred bull calves were used to conduct 3 separate experiments based on age (36 calves/age group: 1 wk of age, 2 mo of age, and 4 mo of age) at the Agriculture and Agri-Food Canada Lethbridge Research Centre (LRC; Lethbridge, AB, Canada). The experiment was conducted as a completely randomized block design in which cattle were blocked by BW, and the treatments were 1) sham castration (CT), where calves were handled in the same manner and for similar amounts of time as castrated calves but not castrated; 2) band castration (BA), with either rubber rings (Elastrator Pliers and Rings, Kane Veterinary Suppliers Ltd., Edmonton, Alberta) for use in newborn calves and 2-mo-old calves or rubber bands (Callicrate Bander, No-Bull Enterprises, St. Francis, KS) for use in 4-mo-old calves; and 3) knife castration (KN), with either a scalpel in newborn calves or a Newberry knife (Syrvet Inc., Waukee, IA) in 2- and 4-mo-old calves. In addition to the removal of the testes, a sterilized emasculator was placed on the spermatic cords for 30 to 60 s to sever and crush the cords and associated blood vessels. All castrations were performed by the same experienced veterinarian. All cow-calf pairs were transported (between April 3 and 8, 2014) approximately 30 km from a local ranch to the LRC feedlot within the first few days after the calves were born. During the experiments calves were housed with their dams in 3 pens (40 × 27 m; 12 cow-calf pairs/pen) with windbreak fencing to the north and west. Within each pen, calves also had access to calf shelters (2.4 × 3.6 × 1.4 m) that were deep bedded with straw. Cows were fed alfalfa grass hay in a round bale feeder ad libitum, whereas calves suckled from their dams but could also consume hay from the bale feeder. Fresh water was provided at all times from a water trough (0.8 × 0.4 × 0.5 m) located in the middle of the pen. Experiments Design and Treatments Experiment 1: Effect of Band and Knife Castration in 1-wk-Old Beef Calves One-week-old calves (4 ± 1.1 d of age, 43 ± 1.1 kg BW) were assessed for indicators of chronic pain until the last remaining scrotum of the BA calves had sloughed off (63 d). Newborn calves were placed on top of large square straw bales and manually restrained during castration while in lateral recumbency. Two calves were removed from the study on d −1 and 7 due to poor health unrelated to castration; consequently, 34 calves were used in Exp. 1: n = 11 for CT, n = 11 for BA, and n = 12 for KN. Experiment 2: Effect of Band and Knife Castration in 2-mo-Old Beef Calves Two-month-old calves (63 ± 2.3 d of age, 92 ± 1.7 kg BW) were assessed for indicators of chronic pain until the last remaining scrotum of the BA calves had sloughed off (49 d). Calves were restrained in a tip table (Calf Roper, Ram-Bull Ltd., Barons, AB, Canada) during castration while in lateral recumbency. Two calves died before the experiment started. In addition, 1 calf was removed from the study on d 14 due to poor health unrelated to castration; consequently, 33 calves were used in Exp. 2: n = 10 for CT, n = 12 for BA, and n = 11 for KN. Experiment 3: Effect of Band and Knife Castration in 4-mo-Old Beef Calves Four-month-old calves (125 ± 4.6 d of age, 160 ± 3.4 kg BW) were assessed for indicators of chronic pain until the last remaining scrotum of the BA calves had sloughed off (42 d). Calves were restrained in a hydraulic squeeze chute (Cattlelac Cattle, Reg Cox Feedmixers Ltd., Lethbridge, AB, Canada) during castration while standing. One calf died before the experiment started. In addition, 1 calf was removed from the study on d 14 due to poor health unrelated to castration; consequently, 34 calves were used in Exp. 3: n = 11 for CT, n = 11 for BA, and n = 12 for KN. Measurements of Chronic Pain and Sample Collection All samples were collected when calves were restrained in the squeeze. Salivary and Hair Cortisol Saliva samples were collected from the oral cavity using a cotton swab to evaluate cortisol concentrations on d −1, immediately before castration (d 0), and weekly thereafter until the end of each experiment. All saliva samples were immediately frozen at −20°C for later analysis of cortisol using an immunoassay technique (Salimetrics Assay Kit, Salimetrics, State College, PA). The intra- and interassay CV were 7.9% and 9.1%, respectively. Hair samples were obtained by shaving the forehead of each calf on d −1 and 35 relative to castration and at the end of the study to measure cortisol concentrations as an indicator of chronic stress. Samples were processed following Moya et al. (2013), and quantification of cortisol was determined using an immunoassay technique (Salimetrics Assay Kit, Salimetrics). The intra- and interassay CV were 6.0% and 11.4%, respectively. Blood Count, Substance P, and Haptoglobin A 6-mL blood sample was collected on d −1, immediately before castration (d 0) and weekly thereafter until the end of each experiment via jugular venipuncture from all calves into a tube containing EDTA (BD Vacutainer, Becton Dickinson Co., Franklin Lakes, NJ) to measure complete blood cell count (CBC) using a HemaTrueHematology Analyzer (Heska, Loveland, CO). Another 6-mL blood sample was collected (according to the same schedule as for CBC) from all calves into a tube containing EDTA (BD Vacutainer, Becton Dickinson Co.) to which benzamidine hydrochloride (1 mM/mL of whole blood) was added for subsequent analysis of substance P concentration (Coetzee et al., 2008). Benzamidine hydrochloride was added to inhibit substance P degradation. The assay was performed at Iowa State University at the College of Veterinary Medicine (Ames, IA) by RIA using a double-antibody system with a primary antibody vs. substance P (1:60,000, H-061-0, Phoenix Pharmaceuticals, Burlingame, CA) and 125I-substance P (20,000 counts/min, PerkinElmer Inc., Waltham, MA) as described by Van Engen et al. (2014). The intra- and interassay CV were 10.7% and 21.0%, respectively. In addition, a 10-mL blood sample was collected on d −1, immediately before castration (d 0) and weekly thereafter until the end of each experiment, using a nonadditive tube (BD Vacutainer, Becton Dickinson Co.) for the analysis of haptoglobin (Hp). Samples were centrifuged at 1,600 × g at 4°C for 15 min, and serum was decanted and stored at −20°C until analysis. Serum Hp concentration was analyzed using a Roche Cobas c501 biochemistry analyzer (Roche Diagnostics, Laval, QC, Canada) using a Tridelta bovine haptoglobin calibrator (TP801CAL, Tridelta, Maynooth, Ireland) and 2 levels of in-house controls (bovine serum pools) daily and 2 levels of Tridelta controls weekly. The interassay CV was 7.6%. Scrotal Area Temperature and Wound Healing Assessment On the same data collection days as the blood samples, thermographic images of the scrotal area were taken using a FLIR i60 infrared camera and processed with ThermCam QuickView 1.3 (Flir systems Inc., Burlington, ON, Canada) to identify potential changes in blood flow and/or inflammation at the scrotal site. The clinical state of the scrotum was scored weekly after castration until the end of each experiment using an 11-point scale (Molony et al., 1995) that was collapsed into the following 5 scores: 0) no swelling, inflammation, or infection visible or palpable; 1) increasing degrees of swelling without obvious erythema; 2) increasing degree of swelling with obvious erythema but without pus; 3) presence of pus with increasing inflammatory response; and 4) presence of suppurative discharge with inflammatory response that required an intervention. Wound healing of the incision state for KN calves was assessed following a 5-point scale (Mintline et al., 2014). Wound healing of BA calves was also assessed using a 5-point scale (Fig. 1). Figure 1. View largeDownload slide Healing score (5-point score) used to evaluate wounds associated with knife (Mintline et al., 2014) and band castration in beef calves. Figure 1. View largeDownload slide Healing score (5-point score) used to evaluate wounds associated with knife (Mintline et al., 2014) and band castration in beef calves. Performance and Rectal Temperature All animals were weighed on 2 consecutive days prior to castration (d −1 and 0) to calculate the initial BW, weekly during the study, and at the end of each experiment (Exp. 1: 68 d postcastration; Exp. 2: 49 d postcastration; Exp. 3: 42 d postcastration). In addition, BW at arrival to LRC and at weaning (October 15, 2014) were recorded for each group. Rectal temperature (GLA M750 Livestock Thermometer, San Luis Obispo, CA) was measured on each day that the calves were weighed. Behavioral Observations Two cameras/pen (2.0MP Bullet Camera, Avigilon, Vancouver, BC, Canada) were mounted on steel poles 6 m above the pens. Cameras continuously recorded the calves from 0800 to 1700 h every day. The video recordings were analyzed with Observer XT software (Noldus Information Technology Inc., Leesburg, VA) using continuous sampling for 2 min every 10 min from 0800 to 1700 h once a week (d 5, 13, 20, and 27 postcastration). An intraclass correlation coefficient with a 95% CI was used to determine interrater and intrarater reliability for 4 different observers who were blind to the treatments. The behaviors evaluated were eating (time [min] calves spent suckling or consuming straw/hay from the ground or feeder), tail flicking (number of times the tail moved from side to side beyond the widest part of the rump), foot stamping (number of times the hind legs were lifted and forcefully placed on the ground or kicked outward while standing), head turning (number of times the head was turned and touched the side of the calf's body, including grooming), and lesion licking (number of times the head was turned to lick the scrotal area). All the behaviors related to pain were scored while animals were standing. Accelerometer Recordings On d −1 all the calves were fitted with an accelerometer data logger (HOBO Pendant G, Onset Computer Corp., Pocasset, MA) to obtain standing and lying time, mean standing and lying duration, and standing and lying bouts (University of British Columbia Animal Welfare Program, 2013). Accelerometers were wrapped with plastic film to protect the device from moisture and foam to eliminate rubbing on the hind leg. Vet Wrap (Professional Preference, Calgary, Canada) was used to strap the accelerometer to the hind leg. The accelerometers were on the calves' legs until d 28 in all the experiments. Stride Length Calf stride length was measured 1 d before (d −1) and immediately after castration on d 0 and weekly thereafter until the end of each experiment. Calves were video recorded (Panasonic WVCP474, Panasonic Canada Inc., Mississauga, ON, Canada) as they traversed a 15-m-long and 1.5-m-wide alley located immediately after the chute. Using video editing software (GOM Media Player, GOM Lab, Gretech Japan Corp., Tokyo, Japan), 2 snapshots were obtained from the video of each calf when both hooves from the back limbs were in contact with the ground. Stride length was calculated as the distance (cm) between both hooves using image editing software (Image J, NIH Image, Bethesda, MD). Calculations and Statistical Analysis Schematic box plots and PROC UNIVARIATE of SAS (version 9.4, SAS Inst. Inc., Cary, NC) were used for outlier detection (none of the data points were removed) and normality testing. Saliva and hair cortisol, substance P, haptoglobin, and CBC were log transformed, and behavioral data were root square +1 transformed to achieve a normal distribution prior to statistical analysis. The individual animal was the experimental unit as treatments were mixed within pen. Performance data were analyzed using a mixed-effects model (SAS version 9.4, SAS Inst. Inc.). The model included castration method as a fixed effect and pen and animal within pen as random effects. Initial BW was used as a covariate. Rectal temperature, maximum scrotal temperature, stride length, and all transformed data described above were analyzed using a mixed-effects model with repeated measures (SAS version 9.4, SAS Inst. Inc.). The model included castration method, time, and their interactions as main effects. Data collected on d −1 and 0 were averaged and used as a covariate with the exception of behavioral data (no baseline precastration behavior data were collected) and stride length (animals were already castrated when stride length was first collected on d 0). Time was considered a repeated factor, and for each analyzed variable, pen and animal within pen (the error term) were subjected to 3 variance-covariance structures: compound symmetry, autoregressive order 1, and unstructured. The covariance structure that minimized Schwarz's Bayesian information criterion was considered the most desirable analysis. The effects of castration method (without including CT) on the time to reduce swelling from the maximum score to no swelling and the time to achieve each healing score were analyzed with a Wilcoxon-Mann-Whitney test (SAS version 9.4, SAS Inst. Inc.). In addition, the univariate procedure (SAS version 9.4, SAS Inst. Inc.) was used to calculate medians and the 95% distribution-free confidence limits. Significance was established at P ≤ 0.05, and trends were indicated at 0.05 < P ≤ 0.10. RESULTS Experiment 1: Effect of Band and Knife Castration on 1-wk-Old Beef Calves Final BW (P = 0.01), weaning BW (P < 0.01), and ADG (P = 0.04) differed among treatments (Table 1). Knife-castrated calves grew less and, consequently, had lower BW at the end of the study (d 68) and at weaning compared to CT and BA calves. No differences (P > 0.10) were observed in salivary or hair cortisol, substance P, haptoglobin, and CBC (white blood cells, red blood cells [RBC], platelets, and neutrophil-to-lymphocyte ratio) among treatments (Table 2). Treatment differences were also not observed for rectal temperature and maximum scrotal temperature (P > 0.10; Table 3). Band-castrated calves had a maximum swelling score of 3 from d 28 to 49, whereas KN calves had a maximum swelling score of 2 from d 7 to 14. A tendency was observed for swelling score 1 (Z = 0.10) and 2 (Z = 0.08), and differences (Z < 0.001) were observed for swelling score 0, with KN calves achieving these scores sooner than BA calves (Table 4). Healing scores of 1, 2, and 5 occurred sooner relative to castration in BA calves (Z < 0.05) compared to KN calves, although no differences were observed when KN or BA calves reached a score of 5 (Table 5). The median day when testicles sloughed off in BA calves was d 56. No differences (P > 0.10) were observed for standing or lying duration or duration of lying bouts among treatments when castration was performed in newborn calves (Table 6). In addition, no chronic effect of castration method (P > 0.10) was observed on stride length, eating time, or behaviors related to pain (tail flicks, foot stamping, head turning, or lesion licking; Table 6) throughout the trial. Table 1. Performance of noncastrated, band-castrated, and knife-castrated Angus crossbred calves castrated at 1 wk, 2 mo, and 4 mo of age from d 7 postcastration until the end of each experiment   Treatment1      Item  CT  BA  KN  SEM  P-value, Trt2  1 wk of age3                Initial BW, kg  41.8  44.5  44.0  1.48  0.39      Final BW, kg  114a  111a  104 b  2.3  0.01      BW weaning, kg  253a  252a  230b  3.9  <0.01      ADG d 68, kg/d  1.04a  1.00a  0.90b  0.034  0.01      ADG weaning, kg/d  1.11a  1.11a  0.99 b  0.021  <0.01  2 mo of age3                Initial BW, kg  91.5  91.7  91.0  1.76  0.96      Final BW, kg  126  120  122  1.9  0.12      BW weaning, kg  236  227  227  4.7  0.28      ADG d 49, kg/d  0.70  0.58  0.62  0.041  0.12      ADG weaning, kg/d  0.77  0.75  0.74  0.027  0.58  4 mo of age3                Initial BW, kg  161  163  162  3.0  0.73      Final BW, kg  206a  202a,b  197b  2.5  0.04      BW weaning, kg  243a  243a  235b  2.6  0.05      ADG d 42, kg/d  1.06a  0.99a,b  0.94b  0.021  0.04      ADG weaning, kg/d  1.14a  1.13a  1.02b  0.037  0.05    Treatment1      Item  CT  BA  KN  SEM  P-value, Trt2  1 wk of age3                Initial BW, kg  41.8  44.5  44.0  1.48  0.39      Final BW, kg  114a  111a  104 b  2.3  0.01      BW weaning, kg  253a  252a  230b  3.9  <0.01      ADG d 68, kg/d  1.04a  1.00a  0.90b  0.034  0.01      ADG weaning, kg/d  1.11a  1.11a  0.99 b  0.021  <0.01  2 mo of age3                Initial BW, kg  91.5  91.7  91.0  1.76  0.96      Final BW, kg  126  120  122  1.9  0.12      BW weaning, kg  236  227  227  4.7  0.28      ADG d 49, kg/d  0.70  0.58  0.62  0.041  0.12      ADG weaning, kg/d  0.77  0.75  0.74  0.027  0.58  4 mo of age3                Initial BW, kg  161  163  162  3.0  0.73      Final BW, kg  206a  202a,b  197b  2.5  0.04      BW weaning, kg  243a  243a  235b  2.6  0.05      ADG d 42, kg/d  1.06a  0.99a,b  0.94b  0.021  0.04      ADG weaning, kg/d  1.14a  1.13a  1.02b  0.037  0.05  a,bWithin rows, values with different superscripts differ (P ≤ 0.05). 1CT: noncastrated but handled as castrated; BA: calves castrated with rubber rings or latex bands; KN: calves castrated with a scalpel or Newberry knife. 2Trt: castration effect. 3Experiment 1: 1-wk-old calves assessed over 68 d; Exp. 2: 2-mo-old calves assessed over 49 d; Exp. 3: 4-mo-old calves assessed over 42 d. Days of study were determined when testicles sloughed off at each age. View Large Table 1. Performance of noncastrated, band-castrated, and knife-castrated Angus crossbred calves castrated at 1 wk, 2 mo, and 4 mo of age from d 7 postcastration until the end of each experiment   Treatment1      Item  CT  BA  KN  SEM  P-value, Trt2  1 wk of age3                Initial BW, kg  41.8  44.5  44.0  1.48  0.39      Final BW, kg  114a  111a  104 b  2.3  0.01      BW weaning, kg  253a  252a  230b  3.9  <0.01      ADG d 68, kg/d  1.04a  1.00a  0.90b  0.034  0.01      ADG weaning, kg/d  1.11a  1.11a  0.99 b  0.021  <0.01  2 mo of age3                Initial BW, kg  91.5  91.7  91.0  1.76  0.96      Final BW, kg  126  120  122  1.9  0.12      BW weaning, kg  236  227  227  4.7  0.28      ADG d 49, kg/d  0.70  0.58  0.62  0.041  0.12      ADG weaning, kg/d  0.77  0.75  0.74  0.027  0.58  4 mo of age3                Initial BW, kg  161  163  162  3.0  0.73      Final BW, kg  206a  202a,b  197b  2.5  0.04      BW weaning, kg  243a  243a  235b  2.6  0.05      ADG d 42, kg/d  1.06a  0.99a,b  0.94b  0.021  0.04      ADG weaning, kg/d  1.14a  1.13a  1.02b  0.037  0.05    Treatment1      Item  CT  BA  KN  SEM  P-value, Trt2  1 wk of age3                Initial BW, kg  41.8  44.5  44.0  1.48  0.39      Final BW, kg  114a  111a  104 b  2.3  0.01      BW weaning, kg  253a  252a  230b  3.9  <0.01      ADG d 68, kg/d  1.04a  1.00a  0.90b  0.034  0.01      ADG weaning, kg/d  1.11a  1.11a  0.99 b  0.021  <0.01  2 mo of age3                Initial BW, kg  91.5  91.7  91.0  1.76  0.96      Final BW, kg  126  120  122  1.9  0.12      BW weaning, kg  236  227  227  4.7  0.28      ADG d 49, kg/d  0.70  0.58  0.62  0.041  0.12      ADG weaning, kg/d  0.77  0.75  0.74  0.027  0.58  4 mo of age3                Initial BW, kg  161  163  162  3.0  0.73      Final BW, kg  206a  202a,b  197b  2.5  0.04      BW weaning, kg  243a  243a  235b  2.6  0.05      ADG d 42, kg/d  1.06a  0.99a,b  0.94b  0.021  0.04      ADG weaning, kg/d  1.14a  1.13a  1.02b  0.037  0.05  a,bWithin rows, values with different superscripts differ (P ≤ 0.05). 1CT: noncastrated but handled as castrated; BA: calves castrated with rubber rings or latex bands; KN: calves castrated with a scalpel or Newberry knife. 2Trt: castration effect. 3Experiment 1: 1-wk-old calves assessed over 68 d; Exp. 2: 2-mo-old calves assessed over 49 d; Exp. 3: 4-mo-old calves assessed over 42 d. Days of study were determined when testicles sloughed off at each age. View Large Table 2. Salivary and hair cortisol, substance P, haptoglobin and blood cell count of uncastrated, band-castrated, and knife-castrated Angus crossbred calves castrated at 1 wk, 2 mo, and 4 mo of age from d 7 postcastration until the end of each experiment   Treatment1    P-value2  Item  CT  BA  KN  SEM  Trt  Time  Trt × time  1 wk of age3                    Salivary cortisol,4 nmol/L  1.97  2.05  1.84  0.098  0.58  <0.001  0.52      Substance P,4 pg/mL  60.6  63.9  62.0  0.10  0.48  <0.001  0.20      Haptoglobin,4 g/L  0.17  0.15  0.18  0.042  0.20  <0.001  0.99      Hair cortisol,4 pg/mL  9.28  8.13  9.63  0.103  0.67  <0.001  0.46      Blood cell count5                        WBC, 103/μL  11.0  10.5  10.6  0.39  0.67  <0.001  0.52          RBC, 103/μL  0.5  9.7  9.2  0.25  0.31  <0.001  0.73          Platelet count, 103/μL  557  564  572  52.6  0.98  0.05  0.56          N:L ratio  0.68  0.56  0.63  0.049  0.24  <0.001  0.52  2 mo of age3                    Salivary cortisol,4 nmol/L  1.88  2.18  1.92  0.091  0.75  0.01  0.45      Substance P,4 pg/mL  80.1  76.2  81.0  0.03  0.80  <0.001  0.23      Haptoglobin,4 g/L  0.20  0.20  0.20  0.064  0.92  0.22  0.27      Hair cortisol,4 pg/mL  5.30  7.15  7.77  0.126  0.15  <0.01  0.81      Blood cell count5                        WBC, 103/μL  11.9  11.3  11.4  0.42  0.61  0.19  0.17          RBC, 103/μL  10.8  10.8  11.0  0.14  0.70  <0.001  <0.01          Platelet count, 103/μL  734  707  738  22.9  0.58  <0.001  0.06          N:L ratio  0.59  0.49  0.54  0.047  0.36  <0.01  0.19  4 mo of age3                    Salivary cortisol4, nmol/L  2.60  2.85  2.43  0.076  0.45  <0.001  0.18      Substance P4, pg/mL  103.3  100.3  100.0  0.05  0.90  0.19  0.83      Haptoglobin4, g/L  0.21  0.20  0.19  0.044  0.93  <0.001  0.77      Hair cortisol4, pg/mL  5.42  4.94  5.51  0.134  0.55  <0.01  0.94      Blood cell count5                        WBC, 103/μL  10.2  10.5  10.4  0.47  0.89  <0.01  0.14          RBC, 103/μL  10.1  10.6  10.4  0.16  0.10  <0.001  0.85          Platelet count, 103/μL  555  584  569  19.4  0.59  <0.01  <0.01          N:L ratio  0.51  0.61  0.55  0.073  0.65  <0.001  0.16    Treatment1    P-value2  Item  CT  BA  KN  SEM  Trt  Time  Trt × time  1 wk of age3                    Salivary cortisol,4 nmol/L  1.97  2.05  1.84  0.098  0.58  <0.001  0.52      Substance P,4 pg/mL  60.6  63.9  62.0  0.10  0.48  <0.001  0.20      Haptoglobin,4 g/L  0.17  0.15  0.18  0.042  0.20  <0.001  0.99      Hair cortisol,4 pg/mL  9.28  8.13  9.63  0.103  0.67  <0.001  0.46      Blood cell count5                        WBC, 103/μL  11.0  10.5  10.6  0.39  0.67  <0.001  0.52          RBC, 103/μL  0.5  9.7  9.2  0.25  0.31  <0.001  0.73          Platelet count, 103/μL  557  564  572  52.6  0.98  0.05  0.56          N:L ratio  0.68  0.56  0.63  0.049  0.24  <0.001  0.52  2 mo of age3                    Salivary cortisol,4 nmol/L  1.88  2.18  1.92  0.091  0.75  0.01  0.45      Substance P,4 pg/mL  80.1  76.2  81.0  0.03  0.80  <0.001  0.23      Haptoglobin,4 g/L  0.20  0.20  0.20  0.064  0.92  0.22  0.27      Hair cortisol,4 pg/mL  5.30  7.15  7.77  0.126  0.15  <0.01  0.81      Blood cell count5                        WBC, 103/μL  11.9  11.3  11.4  0.42  0.61  0.19  0.17          RBC, 103/μL  10.8  10.8  11.0  0.14  0.70  <0.001  <0.01          Platelet count, 103/μL  734  707  738  22.9  0.58  <0.001  0.06          N:L ratio  0.59  0.49  0.54  0.047  0.36  <0.01  0.19  4 mo of age3                    Salivary cortisol4, nmol/L  2.60  2.85  2.43  0.076  0.45  <0.001  0.18      Substance P4, pg/mL  103.3  100.3  100.0  0.05  0.90  0.19  0.83      Haptoglobin4, g/L  0.21  0.20  0.19  0.044  0.93  <0.001  0.77      Hair cortisol4, pg/mL  5.42  4.94  5.51  0.134  0.55  <0.01  0.94      Blood cell count5                        WBC, 103/μL  10.2  10.5  10.4  0.47  0.89  <0.01  0.14          RBC, 103/μL  10.1  10.6  10.4  0.16  0.10  <0.001  0.85          Platelet count, 103/μL  555  584  569  19.4  0.59  <0.01  <0.01          N:L ratio  0.51  0.61  0.55  0.073  0.65  <0.001  0.16  1CT: noncastrated calves handled as castrated; BA: calves castrated with rubber rings or latex bands; KN: calves castrated with a scalpel or Newberry knife. 2Trt: castration effect; Time: time effect; Trt × time: interaction between castration and time effect. 3Experiment 1: 1-wk-old calves assessed over 68 d; Exp. 2: 2-mo-old calves assessed over 49 d; Exp. 3: 4-mo-old calves assessed over 42 d. Days of study were determined when testicles slough off at each age. 4Data transformed to Napierian logarithm. Data presented herein are least squares means without transformation, SEM and P-values from transformed values. 5Blood cell count: WBC: white blood cells; RBC: red blood cells; N:L: neutrophil-to-lymphocyte ratio. View Large Table 2. Salivary and hair cortisol, substance P, haptoglobin and blood cell count of uncastrated, band-castrated, and knife-castrated Angus crossbred calves castrated at 1 wk, 2 mo, and 4 mo of age from d 7 postcastration until the end of each experiment   Treatment1    P-value2  Item  CT  BA  KN  SEM  Trt  Time  Trt × time  1 wk of age3                    Salivary cortisol,4 nmol/L  1.97  2.05  1.84  0.098  0.58  <0.001  0.52      Substance P,4 pg/mL  60.6  63.9  62.0  0.10  0.48  <0.001  0.20      Haptoglobin,4 g/L  0.17  0.15  0.18  0.042  0.20  <0.001  0.99      Hair cortisol,4 pg/mL  9.28  8.13  9.63  0.103  0.67  <0.001  0.46      Blood cell count5                        WBC, 103/μL  11.0  10.5  10.6  0.39  0.67  <0.001  0.52          RBC, 103/μL  0.5  9.7  9.2  0.25  0.31  <0.001  0.73          Platelet count, 103/μL  557  564  572  52.6  0.98  0.05  0.56          N:L ratio  0.68  0.56  0.63  0.049  0.24  <0.001  0.52  2 mo of age3                    Salivary cortisol,4 nmol/L  1.88  2.18  1.92  0.091  0.75  0.01  0.45      Substance P,4 pg/mL  80.1  76.2  81.0  0.03  0.80  <0.001  0.23      Haptoglobin,4 g/L  0.20  0.20  0.20  0.064  0.92  0.22  0.27      Hair cortisol,4 pg/mL  5.30  7.15  7.77  0.126  0.15  <0.01  0.81      Blood cell count5                        WBC, 103/μL  11.9  11.3  11.4  0.42  0.61  0.19  0.17          RBC, 103/μL  10.8  10.8  11.0  0.14  0.70  <0.001  <0.01          Platelet count, 103/μL  734  707  738  22.9  0.58  <0.001  0.06          N:L ratio  0.59  0.49  0.54  0.047  0.36  <0.01  0.19  4 mo of age3                    Salivary cortisol4, nmol/L  2.60  2.85  2.43  0.076  0.45  <0.001  0.18      Substance P4, pg/mL  103.3  100.3  100.0  0.05  0.90  0.19  0.83      Haptoglobin4, g/L  0.21  0.20  0.19  0.044  0.93  <0.001  0.77      Hair cortisol4, pg/mL  5.42  4.94  5.51  0.134  0.55  <0.01  0.94      Blood cell count5                        WBC, 103/μL  10.2  10.5  10.4  0.47  0.89  <0.01  0.14          RBC, 103/μL  10.1  10.6  10.4  0.16  0.10  <0.001  0.85          Platelet count, 103/μL  555  584  569  19.4  0.59  <0.01  <0.01          N:L ratio  0.51  0.61  0.55  0.073  0.65  <0.001  0.16    Treatment1    P-value2  Item  CT  BA  KN  SEM  Trt  Time  Trt × time  1 wk of age3                    Salivary cortisol,4 nmol/L  1.97  2.05  1.84  0.098  0.58  <0.001  0.52      Substance P,4 pg/mL  60.6  63.9  62.0  0.10  0.48  <0.001  0.20      Haptoglobin,4 g/L  0.17  0.15  0.18  0.042  0.20  <0.001  0.99      Hair cortisol,4 pg/mL  9.28  8.13  9.63  0.103  0.67  <0.001  0.46      Blood cell count5                        WBC, 103/μL  11.0  10.5  10.6  0.39  0.67  <0.001  0.52          RBC, 103/μL  0.5  9.7  9.2  0.25  0.31  <0.001  0.73          Platelet count, 103/μL  557  564  572  52.6  0.98  0.05  0.56          N:L ratio  0.68  0.56  0.63  0.049  0.24  <0.001  0.52  2 mo of age3                    Salivary cortisol,4 nmol/L  1.88  2.18  1.92  0.091  0.75  0.01  0.45      Substance P,4 pg/mL  80.1  76.2  81.0  0.03  0.80  <0.001  0.23      Haptoglobin,4 g/L  0.20  0.20  0.20  0.064  0.92  0.22  0.27      Hair cortisol,4 pg/mL  5.30  7.15  7.77  0.126  0.15  <0.01  0.81      Blood cell count5                        WBC, 103/μL  11.9  11.3  11.4  0.42  0.61  0.19  0.17          RBC, 103/μL  10.8  10.8  11.0  0.14  0.70  <0.001  <0.01          Platelet count, 103/μL  734  707  738  22.9  0.58  <0.001  0.06          N:L ratio  0.59  0.49  0.54  0.047  0.36  <0.01  0.19  4 mo of age3                    Salivary cortisol4, nmol/L  2.60  2.85  2.43  0.076  0.45  <0.001  0.18      Substance P4, pg/mL  103.3  100.3  100.0  0.05  0.90  0.19  0.83      Haptoglobin4, g/L  0.21  0.20  0.19  0.044  0.93  <0.001  0.77      Hair cortisol4, pg/mL  5.42  4.94  5.51  0.134  0.55  <0.01  0.94      Blood cell count5                        WBC, 103/μL  10.2  10.5  10.4  0.47  0.89  <0.01  0.14          RBC, 103/μL  10.1  10.6  10.4  0.16  0.10  <0.001  0.85          Platelet count, 103/μL  555  584  569  19.4  0.59  <0.01  <0.01          N:L ratio  0.51  0.61  0.55  0.073  0.65  <0.001  0.16  1CT: noncastrated calves handled as castrated; BA: calves castrated with rubber rings or latex bands; KN: calves castrated with a scalpel or Newberry knife. 2Trt: castration effect; Time: time effect; Trt × time: interaction between castration and time effect. 3Experiment 1: 1-wk-old calves assessed over 68 d; Exp. 2: 2-mo-old calves assessed over 49 d; Exp. 3: 4-mo-old calves assessed over 42 d. Days of study were determined when testicles slough off at each age. 4Data transformed to Napierian logarithm. Data presented herein are least squares means without transformation, SEM and P-values from transformed values. 5Blood cell count: WBC: white blood cells; RBC: red blood cells; N:L: neutrophil-to-lymphocyte ratio. View Large Table 3. Rectal and maximum scrotal temperature of uncastrated, band-castrated, and knife-castrated Angus crossbred calves castrated at 1 wk, 2 mo, and 4 mo of age from d 7 postcastration until the end of each experiment   Treatment1    P-value2  Item  CT  BA  KN  SEM  Trt  Time  Trt × time  1 wk of age3                    Rectal temperature, °C  39.2  39.1  39.2  0.09  0.35  <0.001  0.80      Maximum scrotal temperature, °C  34.9  35.2  35.0  0.28  0.75  <0.001  0.69  2 mo of age3                    Rectal temperature, °C  39.1  39.0  38.4  0.74  0.41  0.23  0.47      Maximum scrotal temperature, °C  37.0  37.1  37.2  0.18  0.77  <0.001  0.23  4 mo of age3                    Rectal temperature, °C  38.7  38.8  38.7  0.11  0.11  <0.001  0.23      Maximum scrotal temperature, °C  36.7b  37.2a  36.6b  0.11  <0.001  <0.001  0.35    Treatment1    P-value2  Item  CT  BA  KN  SEM  Trt  Time  Trt × time  1 wk of age3                    Rectal temperature, °C  39.2  39.1  39.2  0.09  0.35  <0.001  0.80      Maximum scrotal temperature, °C  34.9  35.2  35.0  0.28  0.75  <0.001  0.69  2 mo of age3                    Rectal temperature, °C  39.1  39.0  38.4  0.74  0.41  0.23  0.47      Maximum scrotal temperature, °C  37.0  37.1  37.2  0.18  0.77  <0.001  0.23  4 mo of age3                    Rectal temperature, °C  38.7  38.8  38.7  0.11  0.11  <0.001  0.23      Maximum scrotal temperature, °C  36.7b  37.2a  36.6b  0.11  <0.001  <0.001  0.35  a,bWithin rows, values with different superscripts differ (P ≤ 0.05). 1CT: noncastrated calves handled as castrated; BA: calves castrated with rubber rings or latex bands; KN: calves castrated with a scalpel or Newberry knife. 2Trt: castration effect; Time: time effect; Trt × time: interaction between castration and time effect. 3Experiment 1: 1-wk-old calves assessed over 68 d; Exp. 2: 2-mo-old calves assessed over 49 d; Exp. 3: 4-mo-old calves assessed over 42 d. Days of study were determined when testicles slough off at each age. View Large Table 3. Rectal and maximum scrotal temperature of uncastrated, band-castrated, and knife-castrated Angus crossbred calves castrated at 1 wk, 2 mo, and 4 mo of age from d 7 postcastration until the end of each experiment   Treatment1    P-value2  Item  CT  BA  KN  SEM  Trt  Time  Trt × time  1 wk of age3                    Rectal temperature, °C  39.2  39.1  39.2  0.09  0.35  <0.001  0.80      Maximum scrotal temperature, °C  34.9  35.2  35.0  0.28  0.75  <0.001  0.69  2 mo of age3                    Rectal temperature, °C  39.1  39.0  38.4  0.74  0.41  0.23  0.47      Maximum scrotal temperature, °C  37.0  37.1  37.2  0.18  0.77  <0.001  0.23  4 mo of age3                    Rectal temperature, °C  38.7  38.8  38.7  0.11  0.11  <0.001  0.23      Maximum scrotal temperature, °C  36.7b  37.2a  36.6b  0.11  <0.001  <0.001  0.35    Treatment1    P-value2  Item  CT  BA  KN  SEM  Trt  Time  Trt × time  1 wk of age3                    Rectal temperature, °C  39.2  39.1  39.2  0.09  0.35  <0.001  0.80      Maximum scrotal temperature, °C  34.9  35.2  35.0  0.28  0.75  <0.001  0.69  2 mo of age3                    Rectal temperature, °C  39.1  39.0  38.4  0.74  0.41  0.23  0.47      Maximum scrotal temperature, °C  37.0  37.1  37.2  0.18  0.77  <0.001  0.23  4 mo of age3                    Rectal temperature, °C  38.7  38.8  38.7  0.11  0.11  <0.001  0.23      Maximum scrotal temperature, °C  36.7b  37.2a  36.6b  0.11  <0.001  <0.001  0.35  a,bWithin rows, values with different superscripts differ (P ≤ 0.05). 1CT: noncastrated calves handled as castrated; BA: calves castrated with rubber rings or latex bands; KN: calves castrated with a scalpel or Newberry knife. 2Trt: castration effect; Time: time effect; Trt × time: interaction between castration and time effect. 3Experiment 1: 1-wk-old calves assessed over 68 d; Exp. 2: 2-mo-old calves assessed over 49 d; Exp. 3: 4-mo-old calves assessed over 42 d. Days of study were determined when testicles slough off at each age. View Large Table 4. Swelling score of band- and knife-castrated Angus crossbred calves castrated at 1 wk, 2 mo, and 4 mo of age from d 7 postcastration until the end of each experiment   Treatment1      BA  KN    Score  N  Median  95% FCL3  N  Median  95% FCL3  P-value, Trt2  1 wk of age4      3  3  28  28  —  —  —  —      2  4  14  14–21  2  7  7  0.10      1  10  7  7–49  12  7  7–14  0.08      0  6  49  49–63  12  35  28–42  <0.001  2 mo of age4      4  7  21  21–28  4  7  7  <0.01      3  3  14  14  6  7  7–14  0.41      2  7  14  14–21  3  7  7  0.24      1  12  28  28–42  11  7  7–21  <0.001      0  8  42  35–49  11  35  35–42  <0.001  4 mo of age4      4  11  14  14–21  9  7  7  <0.01      3  6  21  21–28  5  7  7–14  <0.01      2  9  35  28–42  4  14  14  <0.01      1  7  35  35–42  9  21  21  <0.001      0  —  —  —  10  28  28–35  —    Treatment1      BA  KN    Score  N  Median  95% FCL3  N  Median  95% FCL3  P-value, Trt2  1 wk of age4      3  3  28  28  —  —  —  —      2  4  14  14–21  2  7  7  0.10      1  10  7  7–49  12  7  7–14  0.08      0  6  49  49–63  12  35  28–42  <0.001  2 mo of age4      4  7  21  21–28  4  7  7  <0.01      3  3  14  14  6  7  7–14  0.41      2  7  14  14–21  3  7  7  0.24      1  12  28  28–42  11  7  7–21  <0.001      0  8  42  35–49  11  35  35–42  <0.001  4 mo of age4      4  11  14  14–21  9  7  7  <0.01      3  6  21  21–28  5  7  7–14  <0.01      2  9  35  28–42  4  14  14  <0.01      1  7  35  35–42  9  21  21  <0.001      0  —  —  —  10  28  28–35  —  1BA: calves castrated with rubber rings or latex bands; KN: calves castrated with a scalpel or Newberry knife. 2Trt: castration effect. 3FCL: 95% confidence limits distribution free. 4Experiment 1: 1-wk-old calves assessed over 68 d; Exp. 2: 2-mo-old calves assessed over 49 d; Exp. 3: 4-mo-old calves assessed over 42 d. Days of study were determined when testicles slough off at each age. View Large Table 4. Swelling score of band- and knife-castrated Angus crossbred calves castrated at 1 wk, 2 mo, and 4 mo of age from d 7 postcastration until the end of each experiment   Treatment1      BA  KN    Score  N  Median  95% FCL3  N  Median  95% FCL3  P-value, Trt2  1 wk of age4      3  3  28  28  —  —  —  —      2  4  14  14–21  2  7  7  0.10      1  10  7  7–49  12  7  7–14  0.08      0  6  49  49–63  12  35  28–42  <0.001  2 mo of age4      4  7  21  21–28  4  7  7  <0.01      3  3  14  14  6  7  7–14  0.41      2  7  14  14–21  3  7  7  0.24      1  12  28  28–42  11  7  7–21  <0.001      0  8  42  35–49  11  35  35–42  <0.001  4 mo of age4      4  11  14  14–21  9  7  7  <0.01      3  6  21  21–28  5  7  7–14  <0.01      2  9  35  28–42  4  14  14  <0.01      1  7  35  35–42  9  21  21  <0.001      0  —  —  —  10  28  28–35  —    Treatment1      BA  KN    Score  N  Median  95% FCL3  N  Median  95% FCL3  P-value, Trt2  1 wk of age4      3  3  28  28  —  —  —  —      2  4  14  14–21  2  7  7  0.10      1  10  7  7–49  12  7  7–14  0.08      0  6  49  49–63  12  35  28–42  <0.001  2 mo of age4      4  7  21  21–28  4  7  7  <0.01      3  3  14  14  6  7  7–14  0.41      2  7  14  14–21  3  7  7  0.24      1  12  28  28–42  11  7  7–21  <0.001      0  8  42  35–49  11  35  35–42  <0.001  4 mo of age4      4  11  14  14–21  9  7  7  <0.01      3  6  21  21–28  5  7  7–14  <0.01      2  9  35  28–42  4  14  14  <0.01      1  7  35  35–42  9  21  21  <0.001      0  —  —  —  10  28  28–35  —  1BA: calves castrated with rubber rings or latex bands; KN: calves castrated with a scalpel or Newberry knife. 2Trt: castration effect. 3FCL: 95% confidence limits distribution free. 4Experiment 1: 1-wk-old calves assessed over 68 d; Exp. 2: 2-mo-old calves assessed over 49 d; Exp. 3: 4-mo-old calves assessed over 42 d. Days of study were determined when testicles slough off at each age. View Large Table 5. Healing score of band- and knife-castrated Angus crossbred calves castrated at 1 wk, 2 mo, and 4 mo of age from d 7 postcastration until the end of each experiment   Treatment1      BA  KN    Score  N  Median  95% FCL3  N  Median  95% FCL3  P-value, Trt2  1 wk of age4      1  11  7  7  5  7  7–14  0.03      2  9  14  14–21  12  7  7–14  <0.01      3  11  21  14–42  9  28  28–35  0.15      4  12  49  28–68  11  28  28–42  <0.001      5  3  49  49  12  49  42–63  0.41  2 mo of age4      1  12  7  7  —  —  —  —      2  6  14  14  11  7  7  <0.001      3  11  14  14–21  9  21  21–28  <0.01      4  12  28  28–42  11  35  28–42  0.12      5  5  42  42–49  10  49  49  0.16  4 mo of age4      1  11  7  7  —  —  —  —      2  6  14  14  11  7  7  <0.001      3  11  14  14–21  7  28  28  <0.01      4  11  35  28–42  3  29  28  0.05      5  —  —  —  7  35  35  —    Treatment1      BA  KN    Score  N  Median  95% FCL3  N  Median  95% FCL3  P-value, Trt2  1 wk of age4      1  11  7  7  5  7  7–14  0.03      2  9  14  14–21  12  7  7–14  <0.01      3  11  21  14–42  9  28  28–35  0.15      4  12  49  28–68  11  28  28–42  <0.001      5  3  49  49  12  49  42–63  0.41  2 mo of age4      1  12  7  7  —  —  —  —      2  6  14  14  11  7  7  <0.001      3  11  14  14–21  9  21  21–28  <0.01      4  12  28  28–42  11  35  28–42  0.12      5  5  42  42–49  10  49  49  0.16  4 mo of age4      1  11  7  7  —  —  —  —      2  6  14  14  11  7  7  <0.001      3  11  14  14–21  7  28  28  <0.01      4  11  35  28–42  3  29  28  0.05      5  —  —  —  7  35  35  —  1BA: calves castrated with rubber rings or latex bands; KN: calves castrated with a scalpel or Newberry knife. 2Trt: castration effect. 3FCL: 95% confidence limits distribution free. 4Experiment 1: 1-wk-old calves assessed over 68 d; Exp. 2: 2-mo-old calves assessed over 49 d; Experiment 3: 4-mo-old calves assessed over 42 d. Days of study were determined when testicles slough off at each age. View Large Table 5. Healing score of band- and knife-castrated Angus crossbred calves castrated at 1 wk, 2 mo, and 4 mo of age from d 7 postcastration until the end of each experiment   Treatment1      BA  KN    Score  N  Median  95% FCL3  N  Median  95% FCL3  P-value, Trt2  1 wk of age4      1  11  7  7  5  7  7–14  0.03      2  9  14  14–21  12  7  7–14  <0.01      3  11  21  14–42  9  28  28–35  0.15      4  12  49  28–68  11  28  28–42  <0.001      5  3  49  49  12  49  42–63  0.41  2 mo of age4      1  12  7  7  —  —  —  —      2  6  14  14  11  7  7  <0.001      3  11  14  14–21  9  21  21–28  <0.01      4  12  28  28–42  11  35  28–42  0.12      5  5  42  42–49  10  49  49  0.16  4 mo of age4      1  11  7  7  —  —  —  —      2  6  14  14  11  7  7  <0.001      3  11  14  14–21  7  28  28  <0.01      4  11  35  28–42  3  29  28  0.05      5  —  —  —  7  35  35  —    Treatment1      BA  KN    Score  N  Median  95% FCL3  N  Median  95% FCL3  P-value, Trt2  1 wk of age4      1  11  7  7  5  7  7–14  0.03      2  9  14  14–21  12  7  7–14  <0.01      3  11  21  14–42  9  28  28–35  0.15      4  12  49  28–68  11  28  28–42  <0.001      5  3  49  49  12  49  42–63  0.41  2 mo of age4      1  12  7  7  —  —  —  —      2  6  14  14  11  7  7  <0.001      3  11  14  14–21  9  21  21–28  <0.01      4  12  28  28–42  11  35  28–42  0.12      5  5  42  42–49  10  49  49  0.16  4 mo of age4      1  11  7  7  —  —  —  —      2  6  14  14  11  7  7  <0.001      3  11  14  14–21  7  28  28  <0.01      4  11  35  28–42  3  29  28  0.05      5  —  —  —  7  35  35  —  1BA: calves castrated with rubber rings or latex bands; KN: calves castrated with a scalpel or Newberry knife. 2Trt: castration effect. 3FCL: 95% confidence limits distribution free. 4Experiment 1: 1-wk-old calves assessed over 68 d; Exp. 2: 2-mo-old calves assessed over 49 d; Experiment 3: 4-mo-old calves assessed over 42 d. Days of study were determined when testicles slough off at each age. View Large Table 6. Behavior of uncastrated, band-castrated, and knife-castrated Angus crossbred calves castrated at 1 wk, 2 mo, and 4 mo of age from d 7 postcastration until the end of each experiment   Treatment1    P-value2  Item  CT  BA  KN  SEM  Trt  Time  Trt × time  1 wk of age3                    Stride length, cm  35.4  36.1  34.9  0.56  0.37  <0.001  0.69      Lying, %  69.3  69.5  69.4  0.65  0.96  <0.001  0.35      Standing duration, min  27.0  25.1  26.3  1.86  0.76  <0.001  0.64      Lying duration, min  49.6  48.9  51.3  2.04  0.69  <0.001  0.64      Standing bouts, n  20.1  18.6  18.4  0.93  0.35  0.42  0.45      Lying bouts, n  28.6  23.8  22.2  3.47  0.39  0.94  0.41      Eating,4 min  3.8  6.4  5.5  0.30  0.20  0.99  0.91      Tail flick,4 n  17.2  64.6  34.6  0.98  0.15  0.05  0.22      Foot stamp,4 n  17.5  20.1  15.6  0.64  0.93  0.89  0.16      Head turning,4 n  10.9  12.9  9.7  0.22  0.30  0.07  0.44      Lesion licking,4 n  0.51  0.79  0.28  0.15  0.53  0.45  0.79  2 mo of ages3                    Stride length, cm  40.6  41.3  40.8  0.58  0.63  0.01  0.67      Lying, %  55.6  54.2  55.8  0.89  0.39  <0.001  0.72      Standing duration, min  82.7  82.8  81.5  7.74  0.99  <0.001  0.99      Lying duration, min  63.7  61.1  60.3  2.11  0.52  <0.001  0.74      Standing bouts, n  10.4  10.4  9.7  0.29  0.15  <0.001  0.25      Lying bouts, n  13.6  14.0  14.9  0.68  0.38  <0.001  0.21      Eating,4 min  21.5  22.5  23.1  0.29  0.98  <0.01  0.19      Tail flick,4n  77.9  74.4  108.9  0.88  0.62  0.10  0.51      Foot stamp,4n  10.1  10.9  9.5  0.33  0.99  <0.01  0.95      Head turning,4n  12.1  13.1  8.9  0.25  0.24  <0.01  0.38      Lesion licking,4n  0.64  0.80  0.82  0.13  0.27  0.22  0.06  4 mo of age3                    Stride length, cm  46.3  44.2  45.3  0.89  0.26  0.03  0.43      Lying, %  58.1a  53.7b  57.9a  0.63  <0.001  <0.001  0.83      Standing duration, min  68.9b  78.9a  64.6b  1.87  <0.001  <0.001  <0.001      Lying duration, min  58.3b  67.4a  62.8a,b  2.02  <0.01  <0.001  0.33      Standing bouts, n  9.0  9.6  9.2  0.29  0.16  <0.001  0.07      Lying bouts, n  18.0a  12.5b  15.6a,b  0.68  <0.001  <0.001  0.98      Eating,4 min  25.4  22.7  28.3  0.46  0.86  0.70  0.96      Tail flick,4n  13.1  180.8  209.9  1.17  0.38  <0.01  0.08      Foot stamp,4n  10.6  15.1  12.2  0.32  0.25  <0.001  0.33      Head turning,4n  6.9  6.9  10.0  0.26  0.41  <0.01  0.80      Lesion licking,4n  1.1  2.2  1.1  0.17  0.36  0.12  0.33    Treatment1    P-value2  Item  CT  BA  KN  SEM  Trt  Time  Trt × time  1 wk of age3                    Stride length, cm  35.4  36.1  34.9  0.56  0.37  <0.001  0.69      Lying, %  69.3  69.5  69.4  0.65  0.96  <0.001  0.35      Standing duration, min  27.0  25.1  26.3  1.86  0.76  <0.001  0.64      Lying duration, min  49.6  48.9  51.3  2.04  0.69  <0.001  0.64      Standing bouts, n  20.1  18.6  18.4  0.93  0.35  0.42  0.45      Lying bouts, n  28.6  23.8  22.2  3.47  0.39  0.94  0.41      Eating,4 min  3.8  6.4  5.5  0.30  0.20  0.99  0.91      Tail flick,4 n  17.2  64.6  34.6  0.98  0.15  0.05  0.22      Foot stamp,4 n  17.5  20.1  15.6  0.64  0.93  0.89  0.16      Head turning,4 n  10.9  12.9  9.7  0.22  0.30  0.07  0.44      Lesion licking,4 n  0.51  0.79  0.28  0.15  0.53  0.45  0.79  2 mo of ages3                    Stride length, cm  40.6  41.3  40.8  0.58  0.63  0.01  0.67      Lying, %  55.6  54.2  55.8  0.89  0.39  <0.001  0.72      Standing duration, min  82.7  82.8  81.5  7.74  0.99  <0.001  0.99      Lying duration, min  63.7  61.1  60.3  2.11  0.52  <0.001  0.74      Standing bouts, n  10.4  10.4  9.7  0.29  0.15  <0.001  0.25      Lying bouts, n  13.6  14.0  14.9  0.68  0.38  <0.001  0.21      Eating,4 min  21.5  22.5  23.1  0.29  0.98  <0.01  0.19      Tail flick,4n  77.9  74.4  108.9  0.88  0.62  0.10  0.51      Foot stamp,4n  10.1  10.9  9.5  0.33  0.99  <0.01  0.95      Head turning,4n  12.1  13.1  8.9  0.25  0.24  <0.01  0.38      Lesion licking,4n  0.64  0.80  0.82  0.13  0.27  0.22  0.06  4 mo of age3                    Stride length, cm  46.3  44.2  45.3  0.89  0.26  0.03  0.43      Lying, %  58.1a  53.7b  57.9a  0.63  <0.001  <0.001  0.83      Standing duration, min  68.9b  78.9a  64.6b  1.87  <0.001  <0.001  <0.001      Lying duration, min  58.3b  67.4a  62.8a,b  2.02  <0.01  <0.001  0.33      Standing bouts, n  9.0  9.6  9.2  0.29  0.16  <0.001  0.07      Lying bouts, n  18.0a  12.5b  15.6a,b  0.68  <0.001  <0.001  0.98      Eating,4 min  25.4  22.7  28.3  0.46  0.86  0.70  0.96      Tail flick,4n  13.1  180.8  209.9  1.17  0.38  <0.01  0.08      Foot stamp,4n  10.6  15.1  12.2  0.32  0.25  <0.001  0.33      Head turning,4n  6.9  6.9  10.0  0.26  0.41  <0.01  0.80      Lesion licking,4n  1.1  2.2  1.1  0.17  0.36  0.12  0.33  a,bWithin rows, values with different superscripts differ (P ≤ 0.05) 1CT: noncastrated calves but handled as castrated; BA: calves castrated with rubber rings or latex bands; KN: calves castrated with a scalpel or Newberry knife. 2Trt: castration effect; Time: time effect; Trt × time: interaction between castration and time effect. 3Experiment 1: 1-wk-old calves assessed over 68 d; Exp. 2: 2-mo-old calves assessed over 49 d; Exp. 3: 4-mo-old calves assessed over 42 d. Days of study were determined when testicles sloughed off at each age. 4Data transformed to root square + 1. Data presented herein are least squares means without transformation, SEM, and P-values from transformed values. View Large Table 6. Behavior of uncastrated, band-castrated, and knife-castrated Angus crossbred calves castrated at 1 wk, 2 mo, and 4 mo of age from d 7 postcastration until the end of each experiment   Treatment1    P-value2  Item  CT  BA  KN  SEM  Trt  Time  Trt × time  1 wk of age3                    Stride length, cm  35.4  36.1  34.9  0.56  0.37  <0.001  0.69      Lying, %  69.3  69.5  69.4  0.65  0.96  <0.001  0.35      Standing duration, min  27.0  25.1  26.3  1.86  0.76  <0.001  0.64      Lying duration, min  49.6  48.9  51.3  2.04  0.69  <0.001  0.64      Standing bouts, n  20.1  18.6  18.4  0.93  0.35  0.42  0.45      Lying bouts, n  28.6  23.8  22.2  3.47  0.39  0.94  0.41      Eating,4 min  3.8  6.4  5.5  0.30  0.20  0.99  0.91      Tail flick,4 n  17.2  64.6  34.6  0.98  0.15  0.05  0.22      Foot stamp,4 n  17.5  20.1  15.6  0.64  0.93  0.89  0.16      Head turning,4 n  10.9  12.9  9.7  0.22  0.30  0.07  0.44      Lesion licking,4 n  0.51  0.79  0.28  0.15  0.53  0.45  0.79  2 mo of ages3                    Stride length, cm  40.6  41.3  40.8  0.58  0.63  0.01  0.67      Lying, %  55.6  54.2  55.8  0.89  0.39  <0.001  0.72      Standing duration, min  82.7  82.8  81.5  7.74  0.99  <0.001  0.99      Lying duration, min  63.7  61.1  60.3  2.11  0.52  <0.001  0.74      Standing bouts, n  10.4  10.4  9.7  0.29  0.15  <0.001  0.25      Lying bouts, n  13.6  14.0  14.9  0.68  0.38  <0.001  0.21      Eating,4 min  21.5  22.5  23.1  0.29  0.98  <0.01  0.19      Tail flick,4n  77.9  74.4  108.9  0.88  0.62  0.10  0.51      Foot stamp,4n  10.1  10.9  9.5  0.33  0.99  <0.01  0.95      Head turning,4n  12.1  13.1  8.9  0.25  0.24  <0.01  0.38      Lesion licking,4n  0.64  0.80  0.82  0.13  0.27  0.22  0.06  4 mo of age3                    Stride length, cm  46.3  44.2  45.3  0.89  0.26  0.03  0.43      Lying, %  58.1a  53.7b  57.9a  0.63  <0.001  <0.001  0.83      Standing duration, min  68.9b  78.9a  64.6b  1.87  <0.001  <0.001  <0.001      Lying duration, min  58.3b  67.4a  62.8a,b  2.02  <0.01  <0.001  0.33      Standing bouts, n  9.0  9.6  9.2  0.29  0.16  <0.001  0.07      Lying bouts, n  18.0a  12.5b  15.6a,b  0.68  <0.001  <0.001  0.98      Eating,4 min  25.4  22.7  28.3  0.46  0.86  0.70  0.96      Tail flick,4n  13.1  180.8  209.9  1.17  0.38  <0.01  0.08      Foot stamp,4n  10.6  15.1  12.2  0.32  0.25  <0.001  0.33      Head turning,4n  6.9  6.9  10.0  0.26  0.41  <0.01  0.80      Lesion licking,4n  1.1  2.2  1.1  0.17  0.36  0.12  0.33    Treatment1    P-value2  Item  CT  BA  KN  SEM  Trt  Time  Trt × time  1 wk of age3                    Stride length, cm  35.4  36.1  34.9  0.56  0.37  <0.001  0.69      Lying, %  69.3  69.5  69.4  0.65  0.96  <0.001  0.35      Standing duration, min  27.0  25.1  26.3  1.86  0.76  <0.001  0.64      Lying duration, min  49.6  48.9  51.3  2.04  0.69  <0.001  0.64      Standing bouts, n  20.1  18.6  18.4  0.93  0.35  0.42  0.45      Lying bouts, n  28.6  23.8  22.2  3.47  0.39  0.94  0.41      Eating,4 min  3.8  6.4  5.5  0.30  0.20  0.99  0.91      Tail flick,4 n  17.2  64.6  34.6  0.98  0.15  0.05  0.22      Foot stamp,4 n  17.5  20.1  15.6  0.64  0.93  0.89  0.16      Head turning,4 n  10.9  12.9  9.7  0.22  0.30  0.07  0.44      Lesion licking,4 n  0.51  0.79  0.28  0.15  0.53  0.45  0.79  2 mo of ages3                    Stride length, cm  40.6  41.3  40.8  0.58  0.63  0.01  0.67      Lying, %  55.6  54.2  55.8  0.89  0.39  <0.001  0.72      Standing duration, min  82.7  82.8  81.5  7.74  0.99  <0.001  0.99      Lying duration, min  63.7  61.1  60.3  2.11  0.52  <0.001  0.74      Standing bouts, n  10.4  10.4  9.7  0.29  0.15  <0.001  0.25      Lying bouts, n  13.6  14.0  14.9  0.68  0.38  <0.001  0.21      Eating,4 min  21.5  22.5  23.1  0.29  0.98  <0.01  0.19      Tail flick,4n  77.9  74.4  108.9  0.88  0.62  0.10  0.51      Foot stamp,4n  10.1  10.9  9.5  0.33  0.99  <0.01  0.95      Head turning,4n  12.1  13.1  8.9  0.25  0.24  <0.01  0.38      Lesion licking,4n  0.64  0.80  0.82  0.13  0.27  0.22  0.06  4 mo of age3                    Stride length, cm  46.3  44.2  45.3  0.89  0.26  0.03  0.43      Lying, %  58.1a  53.7b  57.9a  0.63  <0.001  <0.001  0.83      Standing duration, min  68.9b  78.9a  64.6b  1.87  <0.001  <0.001  <0.001      Lying duration, min  58.3b  67.4a  62.8a,b  2.02  <0.01  <0.001  0.33      Standing bouts, n  9.0  9.6  9.2  0.29  0.16  <0.001  0.07      Lying bouts, n  18.0a  12.5b  15.6a,b  0.68  <0.001  <0.001  0.98      Eating,4 min  25.4  22.7  28.3  0.46  0.86  0.70  0.96      Tail flick,4n  13.1  180.8  209.9  1.17  0.38  <0.01  0.08      Foot stamp,4n  10.6  15.1  12.2  0.32  0.25  <0.001  0.33      Head turning,4n  6.9  6.9  10.0  0.26  0.41  <0.01  0.80      Lesion licking,4n  1.1  2.2  1.1  0.17  0.36  0.12  0.33  a,bWithin rows, values with different superscripts differ (P ≤ 0.05) 1CT: noncastrated calves but handled as castrated; BA: calves castrated with rubber rings or latex bands; KN: calves castrated with a scalpel or Newberry knife. 2Trt: castration effect; Time: time effect; Trt × time: interaction between castration and time effect. 3Experiment 1: 1-wk-old calves assessed over 68 d; Exp. 2: 2-mo-old calves assessed over 49 d; Exp. 3: 4-mo-old calves assessed over 42 d. Days of study were determined when testicles sloughed off at each age. 4Data transformed to root square + 1. Data presented herein are least squares means without transformation, SEM, and P-values from transformed values. View Large Experiment 2: Effect of Band and Knife Castration in 2-mo-Old Beef Calves No differences (P > 0.10) were observed among treatments on final BW, BW at weaning, and ADG (Table 1). However, CT had numerically greater BW at weaning than BA and KN calves. There was also no effect of castration method (P > 0.10) on salivary cortisol, substance P, haptoglobin, and hair cortisol among treatments (Table 2). An interaction between castration method and time was observed for RBC (P < 0.01) and platelet count (P = 0.06). Knife-castrated calves had greater (P < 0.05) RBC on d 49 compared to BA and CT calves. Band-castrated calves had lower (P < 0.05) platelet counts on d 28 and 49 compared to CT and KN calves (Fig. 2). No differences were observed for rectal temperature (P = 0.47) or maximum scrotal temperature (P = 0.23) among treatments (Table 3). The maximum swelling after castration at 2 mo of age was a score of 4 that was achieved at different (Z < 0.01) times (Table 4). A score of 3 was achieved by KN calves on d 7 and between d 21 and 28 in BA calves. No differences were observed between castration methods when swelling scores of 2 and 3 were achieved. Swelling score reached 0 on d 35 in KN calves and d 42 in BA calves (Z < 0.01). Band-castrated calves had healing scores 2 and 3 from d 14 to 28 (Z < 0.01), whereas KN calves had the same score from d 7 to 35. No differences were observed between castration methods on when calves reached healing scores of 4 and 5 (Table 5). The median day when testicles sloughed off in BA calves was d 35. There was no effect of castration method (P > 0.10) on behavioral data collected with HOBO accelerometers, stride length, or visual observations for eating and behaviors related to pain in Exp. 2 (Table 6). Figure 2. View largeDownload slide Platelet count of uncastrated, band-castrated, and knife-castrated Angus crossbred calves castrated at (A) 2 mo of age and (B) 4 mo of age from d 7 postcastration until d 49 and 42, respectively. Asterisks in each day point denote differences among groups (P ≤ 0.05). Figure 2. View largeDownload slide Platelet count of uncastrated, band-castrated, and knife-castrated Angus crossbred calves castrated at (A) 2 mo of age and (B) 4 mo of age from d 7 postcastration until d 49 and 42, respectively. Asterisks in each day point denote differences among groups (P ≤ 0.05). Experiment 3: Effect of Band and Knife Castration in 4-mo-Old Beef Calves No differences (P > 0.10) were observed between CT and BA for final BW, BW at weaning, ADG, or ADG at weaning. In addition, no effect (P > 0.10) of castration method was observed on final BW or ADG between BA and KN (Table 1). However, KN calves had lower (P = 0.05) BW and ADG at weaning compared to CT and BA calves. There was no chronic effect of castration method (P > 0.10) on salivary and hair cortisol, substance P, or haptoglobin among treatments (Table 2). However, an interaction (P ≤ 0.01) between castration method and time was observed for platelet counts. The KN calves had greater platelet counts on d 7 compared to CT and BA calves, whereas on d 28, their platelet counts were less than those of CT and BA calves (Fig. 2). Castration method affected maximum scrotal temperature (P < 0.001). The BA calves had greater scrotal temperatures than CT and KN calves (Table 3). The maximum swelling score achieved in this age group was 4, and BA calves did not achieve a swelling score of 0 during the study. Castration method affected (Z < 0.001) when swelling scores of 4, 3, 2, and 1 were achieved; BA calves had swelling scores of 4 and 3 on d 14 and 21 and scores of 2 and 1 after d 35, whereas KN calves had swelling scores of 3 and 4 on d 7 and scores of 2 and 1 on d 14 and 21. For KN calves a swelling score of 0 was observed on d 28. Band-castrated calves did not achieve a healing score of 5 during the study. Castration method had an effect (Z < 0.001) on day when healing scores of 2, 3, and 4 were achieved: BA calves had healing scores of 2 on d 7, whereas KN calves had healing scores of 2 on d 14, and BA calves had healing scores of 3 and 4 from d 14 to d 42, whereas the same scores where achieved in KN calves on d 28. Knife-castrated calves were completely healed (score 5) on d 35. The median day when testicles sloughed off in BA calves was d 35. No differences (P = 0.43) were observed in stride length among treatments (Table 6). Behavioral data collected from HOBO data accelerometers over 28 d postcastration (Table 6) showed that lying time differed between treatments. The BA calves spent less time lying (P < 0.001) than CT and KN calves, although BA calves had greater average lying times (P < 0.05) than CT calves. The same pattern for lying duration was observed for lying bouts, where BA had fewer (P < 0.001) lying bouts than CT calves. No differences (P > 0.10) between BA and KN were observed for lying duration or lying bouts. An interaction between castration method and time (P < 0.001) was observed for standing duration among treatments (Fig. 3). The BA calves had greater (P < 0.05) standing durations between d 15 and 19 than CT and KN calves. There tended to be an interaction (P < 0.10) between castration method and time for tail flicking; KN calves had greater tail flicking compared CT and BA calves on d 5, whereas no differences among treatments were observed on d 13, 20, and 27 postcastration. Figure 3. View largeDownload slide Standing duration of uncastrated, band-castrated, and knife-castrated Angus crossbred calves castrated at 4 mo of age from d 7 postcastration until d 42. Asterisks in each day point denote differences among groups (P ≤ 0.05). Figure 3. View largeDownload slide Standing duration of uncastrated, band-castrated, and knife-castrated Angus crossbred calves castrated at 4 mo of age from d 7 postcastration until d 42. Asterisks in each day point denote differences among groups (P ≤ 0.05). DISCUSSION Several studies have demonstrated that castration causes acute pain at the time of the procedure and for hours afterward, as evidenced by changes in physiological and behavioral parameters (Robertson et al., 1994; Molony et al., 1995; Fisher et al., 1996; Stafford et al., 2002; Ting et al., 2003; Schwartzkopf-Genswein et al., 2005; Thüer et al., 2007, González et al., 2010). In the present study, chronic pain was evaluated from d 7 postcastration until the testicles of all band castrated calves had sloughed off. Some of the physiological parameters used to assess acute pain (e.g., serum or salivary cortisol) may not be appropriate for assessing chronic pain (Fox, 2009), and other parameters such as clinical-pathological indices should be included to better assess a chronic condition (Molony et al., 1995). Reduced weight gains after castration could be due to a combination of pain (Weary et al., 2006; Pang et al., 2008) and the lack of testosterone (Purchas et al., 2002). However, Bretschneider (2005) reported that growth suppression after castration increased quadratically with age, indicating that the weaning weight of calves castrated close to birth should not differ from that of noncastrated calves. Similar findings were found in the present study in BA calves. However, 1-wk- and 4-mo-old KN calves had a 9% and 3.3% reduction in weaning compared to CT, respectively. In contrast, Brown et al. (2015) did not observe differences in BW at weaning between surgically castrated calves near birth and bulls. Lents et al. (2006) did not find differences at weaning between 2- and 3-mo-old calves surgically and band castrated or kept intact, even though all calves had been implanted with zeranol. Therefore, the reduction of weight for 1-wk- and 4-mo-old surgically castrated calves may indicate that those calves experience chronic stress that was not detected with the other physiological and behavioral parameters measured in the present study. The lack of differences in weaning weight when castration was performed at 2 mo of age, with the low ADG observed in all treatments, could possibly be explained by rumen development. Although 2-mo-old calves still have a milk-based diet, they start to eat forage at this age. Subsequent rumen development changes the bacterial population and promotes growth of the muscular layer of the rumen and papillae (Castells et al., 2013). Subjecting calves to a substantial amount of handling at this time of transition may impair growth regardless of whether the calves were bulls or steers. Further research would be required to confirm this hypothesis. Previous studies indicated that knife castration in newborn (Robertson et al., 1994; Molony et al., 1995), 1- to 2-mo-old (Robertson et al., 1994; Thüer et al., 2007), and 2- to 4-mo-old calves (Stafford et al., 2002) produced greater mean concentrations of cortisol than BA castration during the first 180 min to 72 h after the procedure. In addition, knife-castrated calves also had cortisol peaks 2 to 5 times greater than those of noncastrated calves, depending on the age (Robertson et al., 1994). The lack of significance in salivary cortisol measurements in the current study confirms that salivary cortisol may be a less valuable indicator of chronic pain/stress than for acute pain/stress. Recently, hair cortisol has been used for the assessment of long-term or chronic stress (Davenport et al., 2006; Moya et al., 2013; Tallo-Parra et al., 2017). As hair grows, it incorporates hormones and other substances (Meyer and Novak, 2012), reflecting the state of the animal over time. Results of the present study did not show long-term pain or stress as hair cortisol was similar among castration methods in all ages. Tallo-Parra et al. (2017) showed that hair cortisol was not affected after 2 ACTH challenges simulating an acute stress similar to that produced during castration in the present study. The results of Tallo-Parra et al. (2017) suggest that the acute pain experienced by calves in the present study associated with castration was not intense or long enough to be deposited in great enough quantities in the hair of those calves to observe treatment differences. Substance P is a neuropeptide in the central nervous system associated with stress regulation, anxiety-related behaviors, and pain (Ebner and Singewald, 2006). Substance P concentrations in the present study did not differ among treatments in any age group for the duration of the experiment. Coetzee et al. (2008) observed an increase in substance P in 4- to 6-mo-old knife-castrated calves 240 min after castration, and Mintline et al. (2014) found that substance P concentrations decreased 3 d after castration. The contrasting results between the present study and those of Coetzee et al. (2008) and Mintline et al. (2014) might suggest that substance P would be a better indicator for acute pain/stress than for chronic pain/stress that is associated with castration. The results for substance P in this study should be viewed with caution because of the elevated interassay CV. Plasma concentrations of acute-phase proteins like haptoglobin increase as part of the inflammatory cascade associated with tissue damage or in response to a stressor (Baumann and Gauldie, 1994). Therefore, independent of the castration method, haptoglobin would be expected to increase as local tissue trauma was produced. However, although some swelling was observed in the present study, it was not enough to increase haptoglobin concentrations at any point in time, in any of the calf ages assessed. In contrast, Ting et al. (2004) reported an increase in haptoglobin concentration on the third day after burdizzo castration in 2.5- to 3.5-mo-old calves. Several authors have also reported differences in haptoglobin concentration after castration in weaned calves. Brown et al. (2015) observed an increase in haptoglobin concentration for 72 h after knife castration, and Warnock et al. (2012) showed an increase in haptoglobin concentration in banded calves 15 d after castration, indicating a delayed inflammatory response. The lack of differences in the present study may be due to the age of the calves as Brown et al. (2015) and Warnock et al. (2012) found increases in haptoglobin in weaned calves and the days when haptoglobin samples were collected as differences were seen after 72 h (Brown et al. 2015) and 15 d (Warnock et al., 2012) after castration. Even though haptoglobin concentrations did not increase in the present study, there was evidence of a delayed inflammatory response (swelling score) in BA calves. Both 1-wk- and 2-mo-old calves had inflammation lasting 7 d after knife castration, whereas inflammation was observed for up to 14 d in 4-mo-old calves. Swelling in BA calves lasted for 21 to 28 d in the 2 younger groups of calves, whereas in 4-mo-old calves swelling was observed until d 35. These results are in agreement with those of Molony et al. (1995) and Stafford (2007), who reported knife castration wounds healed at least a week sooner than those in banded calves. We observed that it took longer for the scrotum to slough off and for the remaining lesion to heal in younger calves. This may be explained by the fact that band tension most likely increases as testicle size increases (with age). In the present study, the rubber rings applied on 1-wk- and 2-mo-old calves had a specific diameter, and therefore, the degree of tension exerted would depend on the diameter of the neck of the scrotum. The neck of the scrotum is thinner in younger calves, so more tension is needed to constrict the scrotal tissue to produce ischemia and necrosis. Consequently, when insufficient tension is applied, it takes longer for the testicles to slough off. This was evident in the mean length of time calves at 1 wk and 2 mo of age took to lose their testicles (68 and 49 d, respectively). When band castration was performed in 4-mo-old calves, the latex bands used required the operator to mechanically tighten the band to a particular tension. When the tension is applied according to the diameter of the neck of the scrotum, it allows the band to produce ischemia and necrosis faster; thus, sloughing off the testicles would occur sooner (42 d in the present study). Platelets are known to be involved in all the stages of tissue repair, including coagulation, immune cell recruitment and inflammation, and wound healing, angiogenesis, and remodeling (Golebiewska and Poole, 2015). The changes in the concentration of platelets in the present study indicate how wound healing in BA calves differs among ages. Although no differences were observed in calves castrated at 1 wk of age, the platelet count in 1-wk- and 2-mo-old BA calves decreased when most of the wound was a scab (d 49 and 28 for 1-wk- and 2-mo-old calves, respectively) and peaked again when the scrotum was detaching from the ventral area (d 63 and 42 for 1-wk- and 2-mo-old calves, respectively). In 4-mo-old calves, platelet count remained high until d 35, and on d 42 platelet count decreased, which may be explained by the open wound created in BA calves at older ages, which takes longer to form a scab. Few studies have evaluated the effect of castration on behavior for extended periods of time. Thüer et al. (2007) and Marti et al. (2010) evaluated behavior for 84 and 49 d, respectively, after band castration, and Molony et al. (1995) evaluated behavior for 48 d after band and surgical castration. No statistical differences were observed in most of the behaviors evaluated by Molony et al. (1995); an increase of lesion licking was observed in calves castrated with rubber rings during the 48-d period evaluated in calves castrated at 5 to7 d of age. In the present study, calves castrated at 1 wk or 2 mo of age did not show behaviors related to pain during the 27-d evaluation period, perhaps because the 27-d behavioral observation period ended before the time points when BA calves would show changes in behavior (when testes slough off). However, the fact that testicles at those ages were less developed and less vascularized than in older calves may also have contributed to the lack of behavioral differences, as 4-mo-old BA calves did show changes in behavior compared to CT calves. Results from the present study indicate that physiological, behavioral, and clinical-pathological indicators of pain/stress are significantly influenced by both the method and age of the calves when the castrations were performed. It should be noted that statistical comparisons between age differences could not be done, as data collection of those parameters was done at different time points and using different handling procedures. In addition, welfare indicators of pain and distress such as salivary and hair cortisol, substance P, and haptoglobin were unaffected in the long term after castration independent of method and age. However, behavioral parameters and clinical-pathological evaluations showed that band castration in 4-mo-old calves causes chronic pain lasting 21 d. Meléndez et al. (2017) found that regardless of method and age castration causes acute pain. Therefore, the combined acute and chronic assessments of knife and band castration methods suggest that pain mitigation should be used to improve animal welfare, particularly when performed in calves older than 2 mo of age. However, when access to pain mitigation is limited, band castration in calves younger than 2 mo of age could be used as a strategy to reduce pain/stress. Further research is needed to establish welfare- and producer-friendly pain mitigation strategies for beef calves. LITERATURE CITED Baumann H. Gauldie J. 1994. The acute phase response. Immunol. Today  15: 74– 80. doi: https://doi.org/10.1016/0167-5699(94)90137-6 Bretschneider G 2005. Effects of age and method of castration on performance and stress response of beef male cattle: A review. Livest. Prod. Sci.  97: 89– 100. doi: https://doi.org/10.1016/j.livprodsci.2005.04.006 Google Scholar CrossRef Search ADS   Brown A. C. Powell J. G. Kegley E. B. Gadberry M. S. Reynolds J. L. Hughes H. D. Carroll J. A. 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TI - Effect of band and knife castration of beef calves on welfare indicators of pain at three relevant industry ages: II. Chronic pain
JF - Journal of Animal Science
DO - 10.2527/jas2017.1763
DA - 2017-10-01
UR - https://www.deepdyve.com/lp/oxford-university-press/effect-of-band-and-knife-castration-of-beef-calves-on-welfare-8UujnB86rb
SP - 4367
EP - 4380
VL - 95
IS - 10
DP - DeepDyve
ER -