TY - JOUR
AU - McGowan, M.
AB - ABSTRACT The welfare outcomes for Bos indicus cattle (100 heifers and 50 cows) spayed by either the dropped ovary technique (DOT) or ovariectomy via flank laparotomy (FL) were compared with cattle subjected to physical restraint (PR), restraint by electroimmobilization in conjunction with PR (EIM), and PR and mock AI (MAI). Welfare assessment used measures of morbidity, mortality, BW change, and behavior and physiology indicative of pain and stress. One FL heifer died at d 5 from peritonitis. In the 8-h period postprocedures, plasma bound cortisol concentrations of FL, DOT, and EIM cows were not different and were greater (P < 0.05) than PR and MAI. Similarly, FL and DOT heifers had greater (P < 0.05) concentrations than PR and MAI, with EIM intermediate. Creatine kinase and aspartate aminotransferase concentrations were greater (P < 0.05) in FL and EIM heifers compared with the other treatments, with a similar pattern seen in the cows. Haptoglobin concentrations were significantly (P < 0.05) increased in the FL heifers compared with other treatments in the 8- to 24-h and 24- to 96-h periods postprocedures, and in cows were significantly (P < 0.05) increased in the FL and DOT compared with PR in the 24- to 96-h period. Behavioral responses complemented the physiological responses; standing head down was shown by more (P < 0.05) FL cows and heifers to 3 d postprocedures compared with other treatments, although there was no difference between FL and DOT heifers at the end of the day of procedures. At this same time, fewer (P < 0.05) FL and DOT heifers and cows were observed feeding compared with other treatments, although in cows there was no difference between FL, DOT, and EIM. There were no significant differences (P > 0.05) between treatments in BW changes. For both heifers and cows, FL and DOT spaying caused similar levels of acute pain, but FL had longer-lasting adverse impacts on welfare. Electroimmobilization during FL contributed to the pain and stress of the procedure. We conclude that: i) FL and DOT spaying should not be conducted without measures to manage the associated pain and stress; ii) DOT spaying is preferable to FL spaying; iii) spaying heifers is preferable to spaying cows; and iv) electroimmobilization causes pain and stress and should not be routinely used as a method of restraint. INTRODUCTION In rangeland beef cattle production systems, there are sound economic and welfare reasons for rendering surplus female cattle, particularly heifers and aged breeding females, sterile by spaying; female breeder mortalities are reduced and producers can fatten cull and surplus females within the breeding herd, reducing the need for additional gathering and handling of cattle (Pinner, 2006). Further, spaying of surplus females is reported to be an important method for managing stocking rates to control land degradation (Jubb and Letchford, 1997). Spaying is conducted on extensively managed cattle in northern Australia, North and South America, and southern Africa because there are no other reliable and practical methods for rendering females sterile. Chemical contraceptives, such as deslorelin implants, can be effective for periods of 200 to 300 d (D'Occhio et al., 2002), but in Australia at least, such implants are registered only for companion animals and are significantly more expensive than spaying. Preventing bulls from accessing cull and surplus females in northern Australia is problematic because bulls are kept with females continuously in the majority of herds, paddocks are very large due to low stocking rates, and fences are frequently breached from seasonal floods and bushfires (Petherick, 2005). Traditionally, spaying has been conducted via a flank laparotomy (FL) with or without the use of anesthetics or analgesics. A per-vagina method of spaying, the dropped ovary technique (DOT; Habermehl, 1993) was introduced into northern Australia in 1996 (Jubb et al., 2003) and the uptake was rapid. Although conducted without use of anesthetics or analgesics, DOT spaying is perceived by cattle producers, veterinarians, and animal welfare organizations to offer welfare advantages over FL spaying; however, a rigorous comparison of the impact on welfare of each method has not been conducted (Pinner, 2006) and was, thus, the primary objective of this study. MATERIALS AND METHODS The care and management of the cattle were approved by an Animal Ethics Committee at Charles Darwin University, Darwin, Northern Territory, Australia (approval number A06007) in compliance with state and national legislation. Location and Environmental Conditions The experiment was conducted on a commercial beef cattle property located approximately 500 km southwest of Katherine in the southern portion of the Victoria River District, Northern Territory, Australia (17°12′ S, 130°38′ E). This study was conducted during the dry season (May to July) when cattle spaying is routinely conducted; no rainfall was recorded, the average relative humidity ranged between 32 and 59%, and the minimum and maximum temperature ranges were 6 to 15°C and 20 to 30°C, respectively. Three paddocks were used during the study: Paddock 1 (84.8 ha), Paddock 2 (16.3 ha), and Paddock 3 (21.5 ha). Native grass pastures were the main grazing resource, with the predominant species being Aristida latifolia, Dicanthium fecundum, Chrysopogan fallax, Sesbania cannabina, Astrebla spp., Indigophera spp., and Sorghum intrans. Some paddocks had previously been grazed, and therefore pastures had been used to varying degrees before commencement of the study: Paddock 1: 50% used, estimated 1,500 to 2,000 kg DM/ha yield; Paddock 2: 75 to 100% used, 500 to 800 kg DM/ha yield; and Paddock 3: <10% used, 2,500 to 3,000 kg DM/ha. Cattle held in paddock 2 were supplemented with pasture hay ad libitum. Animals, Treatments, and Experimental Design and Conduct The work was conducted in a set of steel commercial cattle yards and surrounding barbed-wire fenced holding paddocks. Cattle were mustered (gathered) to the yards either using motorbikes or on horseback. Three days before allocation, 123 Brahman cows (aged 2 to 15 yr) and 115 yearling Brahman heifers were mustered, and pregnancy and lactation status, BW, and flight speed were recorded. Flight speed is the speed at which animals traverse a known distance when released from close confinement, such as a weighing crate (Burrow et al., 1988). Two heifers had been dehorned 2 to 3 wk before allocation to the study. The cattle had been identified for spaying by the cooperating cattle company and were, thus, typical of the cattle normally spayed in this region. The data were subjected to a principal components analysis and animals then blocked according to BW and flight speed. Flight speed was considered an important consideration for allocation, as cattle with fast speeds appear more susceptible to stressors, and flight speed is negatively correlated with BW gain (Petherick et al., 2002, 2009). Fifty Brahman cows (439.4 ± 14.5 kg; 12 were 1 to 4 mo pregnant and the others were not detectably pregnant) and 100 non-detectably pregnant, yearling Brahman heifers (208.3 ± 4.3 kg) were selected. Early pregnant cows were included, as both early pregnant and nonpregnant cows are routinely spayed. Within blocks, animals were randomly allocated to 1 of 5 treatment groups, and then grouped into 10 replicates (heifers) or 5 replicates (cows). Each animal was ear-tagged (left ear) with a numbered, colored tag for individual identification. The tag color denoted the treatment, which assisted with the sorting of animals for treatment. Neither the distribution to treatments of cow age (F = 1.1; P = 0.36) nor cow pregnancy status (χ2= 1.75; P = 0.78) was significantly different. All of the manipulative and surgical procedures were carried out without the use of local anesthetics, consistent with industry practice and state regulations. All procedures were carried out by a highly-skilled, experienced veterinarian who typically performs the DOT on 20,000 cattle and FL spaying on 5,000 cattle per annum. There were 5 treatment groups with 20 heifers and 10 cows assigned to each of the treatments. The treatments were used to allow a comparison of FL and DOT spaying methods and to compare them with other management procedures that, under Australian rangeland conditions, are usually a component of these spaying procedures. Spaying using the DOT involves rectal palpation, manipulation of the reproductive tract, and insertion of a device into the vagina, therefore, we examined the responses of cattle to mock AI. Cattle that are spayed using the FL technique are commonly electroimmobilized, and thus we examined the responses of cattle that were electroimmobilized. Physical Restraint (PR). Each animal was physically restrained in a commercial cattle squeeze-chute with the head caught in a parallel closing head-gate and the parallel side-panel squeeze applied for the period of blood sampling and for 1 min afterwards. Dropped Ovary Technique Spaying (DOT). The cattle were physically restrained, as described above and with the kick-gate closed behind the back legs. Cattle were DOT spayed according to procedures described by Jubb et al. (2003) and de Witte et al. (2006). After wiping the vulva clean, the disinfected ovariotome (Willis Spay Tool, Otto Fix Pty. Ltd., Underwood, QLD, Australia) was introduced into the vagina, inserted through the vaginal fornix into the caudal abdominal cavity. Then each ovary was manipulated into the cutting slot of the ovariotome by transrectal palpation and severed. Immediately after the spay procedure was completed, a 15-mm-diam. hole was punched, using standard ear-punch pliers, through the pinna of the left ear, in accordance with state legislation. The entire procedure was completed within 2 min. Mock Artificial Insemination (MAI). The cattle were physically restrained with a kick gate closed behind the back legs. After wiping the vulva clean, an AI gun (D201, IMV Technologies, L'Aigle, France) was inserted into the vagina and manipulated by transrectal palpation of the cervix to enable passage of the tip of the gun through the cervix to the body of the uterus. The entire procedure took approximately 1 min to perform. Flank Laparotomy Spaying (FL). The procedure was performed as described in detail by McCosker et al. (2010). Briefly, the cattle were physically restrained and electoimmobilized, and, after skin disinfection, a 12- to 15-cm incision was made through the left, caudal paralumbar fossa to enable insertion of the hand and arm of the operator. Each ovary was located, severed with a double scalpel mounted cutting instrument (Speymate 23, Hayes Veterinary Supplies, Capalaba, QLD, Australia) and then removed. The flank incision was then closed with a simple continuous suture. The animal was ear punched as previously described, and Cetrigen wound spray [Virbac (Australia) Pty. Ltd., Milperra, NSW, Australia] was applied to the wound because the cattle were regularly handled during the first 24 h after the procedure. On average, the entire procedure was completed in approximately 3 min. Electroimmobilization Restraint (EIM). This procedure is described in detail by McCosker et al. (2010). Briefly, the cattle were physically restrained as previously described and then the 2 electrodes of the “Stockstill” immobilizer unit (Stockstill Ltd, Southfield, SA, Australia) were inserted into the upper lip and the rump/tail-base area, on the same side of the animal. The unit was turned on and the amperage adjusted to cause skeletal muscles to contract, resulting in immobilization but still allowing the animal to breathe. The animals were electoimmobilized for approximately 1 min. Five replicates, each of 10 heifers (2 of each treatment), 5 replicates each of 10 cows (2 of each treatment) and a further 5 replicates each of 10 heifers (2 of each treatment) were treated on 3 successive days. This design was selected to ensure that, on the day that procedures were conducted (d 0), all blood samples could be collected during daylight hours and that there would be sufficient time after the last blood sample for a period of uninterrupted observation of the behaviors of the cattle. On d 0, the cattle within each replicate group were moved into a squeeze-chute and restrained for blood sampling. The treatment, which had been previously assigned to the individual animal, was then immediately conducted and the animal released to a yard. Once all cattle within the replicate had been sampled and treated, the group was moved to another yard to make way for the next replicate group. Groups were moved through a series of yards to return them to a squeeze-chute (either the original, or an identical alternative chute installed in an adjacent yard) for repeated blood sampling, with group movements timed and coordinated to avoid clashes, and to ensure treatments were performed and blood samples taken at scheduled times. Food was not provided in the yards through which the replicate groups were moved, but water was available in some of them. The procedures were repeated until all 5 replicates had been completed. Once all samples had been collected on d 0, the cattle were put together in a large yard, with ad libitum access to water and good quality pasture hay, and held there overnight. The next morning (d 1), the cattle were moved through the yards, individually restrained, and a blood sample taken (approximately 24 h posttreatment). They were then released to 1 of the 3 paddocks adjacent to the yards, with ad libitum pasture, hay and water. All of these procedures were repeated a further 2 times, so that there was a day-replicate herd in each of the 3 paddocks. Three days after treatment (d 4), the cattle were mustered from the paddock and again moved through the yards and restrained for blood sampling. After blood sampling on d 4, the 3 herds were placed together in a single paddock (paddock 1 described previously) and mustered from this paddock, on average, on d 21 and 42 posttreatment for weighing and scoring of wounds. Blood Sampling and Assaying Approximately 10 mL of blood were collected by jugular venipuncture into labeled lithium heparin (for plasma) or plain (for serum) Vacutainer tubes (Becton Dickinson, Plymouth, UK) immediately after the cattle were head-bailed. Blood sampling was scheduled for before treatment (time 0) and 1, 2, 3, 4, 6, 8, 24 and 96 h posttreatment, with the actual time of posttreatment sampling ranging between 0.7 to 1.3, 1.7 to 2.3, 2.7 to 3.4, 3.8 to 4.3, 5.7 to 6.4, 7.2 to 8.2, 21.8 to 24.4, and 95.2 to 97.6 h for these target times, respectively. Blood samples were held at <10oC in a portable refrigerator and, within 1 h of collection, were centrifuged at 1,500 × g for 20 min at room temperature (GS200 centrifuge, Clements Medical Equipment, North Sydney, NSW, Australia) and the plasma/serum samples were decanted into duplicate, labeled, 5-mL, screw-capped storage tubes (Sarstedt Australia Pty. Ltd., Adelaide, Australia) and frozen at –20°C until assay. Plasma cortisol concentrations have been widely used as an indicator of pain associated with invasive husbandry procedures (Mellor et al., 2000); thus, bound cortisol concentrations were determined via ELISA (Cortisol ELISA EIA Saliva kit; DSL Laboratories, Melbourne, Australia) on samples collected at 0, 1, 2, 3, 4, 6, 8, 24 and 96 h and unbound, or free, cortisol concentrations were measured in filtered plasma samples (Sartorius 10kDa MWCO filter, Sartorius, Goettingen, Germany) collected at 0, 8, 24 and 96 h using the same ELISA EIA kit. Plasma concentrations of aspartate aminotransferase (AST), creatine kinase (CK), and NEFA were analyzed with an Olympus Reply Chemistry Analyzer (Olympus, Tokyo, Japan) on blood collected at 0, 8, 24 and 96 h postprocedures. Concentrations of NEFA are indicative of a sympatho-adrenalmeduallary stress response (Blum et al., 1982), whereas CK and AST are indicators of muscle damage, physical exertion, stress and fatigue in cattle (Braun et al., 1993; Garcia-Belenguer et al., 1996; Knowles and Warriss, 2007). Additionally, concentrations of serum haptoglobin, which is an acute-phase protein indicative of acute inflammatory conditions (Horadagoda et al., 1999), were measured in blood samples collected at 0, 24, and 96 h postprocedures using the Olympus Reply Chemistry Analyzer (Olympus, Tokyo, Japan). Behavioral Recordings An ethogram of mutually exclusive behaviors (Table 1) was developed by a cattle behavior expert (J. C. Petherick). Behavioral observations were made by this expert on the cattle on d 0, both when in the replicate groups and when grouped together at the end of the day. Observations of the cattle in the paddocks (d 1 to 3) were conducted by this same observer and 2 other cattle experts who had been trained by the expert observer, using the ethogram, to standardize recordings. The observers were blinded to the treatments applied, although it was evident which cattle were on the FL treatment because of the sutured wound in their flank. The behavioral recordings were classified into 3 time periods according to when and where they were made: i) on d 0 between blood-sampling times when the cattle were in their replicate groups; ii) at the end of d 0 when the 5 replicate groups were co-mingled in a large yard; and iii) on d 1 to 3 when the 3 groups were in the 3 paddocks. Table 1. Ethogram developed for investigating changes in the behavior of heifers and cows in response to restraint, mock AI, electroimmobilization, spaying by the dropped ovary technique, and flank spaying with electroimmobilization Behavior  Description  Standing head down  Head level with or below brisket  Standing head up  Head above brisket  Standing stiff-tailed  Standing with tail held stiffly away from body  Lying sternal recumbency  Lying on sternum or partially on sternum with hind-quarters to one side  Lying lateral recumbency  Lying on side, fully-recumbent  Locomotion  Walking, trotting  Feeding  Taking hay into mouth and/or chewing hay and/or grazing and/or browsing  Drinking  Consuming water  Ruminating standing  Standing, generally with a relaxed posture with regular chewing and regurgitation movements  Ruminating lying  As above, but lying sternum  Licking standing/lying  Standing or lying on sternum, turning to lick or attempt to lick body (body region noted)  Rub/scratch  Rubbing/scratching head or body against an object  Vocalization  Bellow or low  Teeth-grinding  Grinding molars together  Shiver/tremble  Whole of body shivering, shaking or trembling  Butt  Butt or attempted butt directed at another animal  Charge  Charges at another animal and stops  Push  Pushes another animal out of the way  Chase  Chases another animal (pursuit continues for some seconds)  Retreat  Moves away from butt, charge, push or chase  Grooms another  Licks another animal  Receives grooming  Recipient of grooming  Behavior  Description  Standing head down  Head level with or below brisket  Standing head up  Head above brisket  Standing stiff-tailed  Standing with tail held stiffly away from body  Lying sternal recumbency  Lying on sternum or partially on sternum with hind-quarters to one side  Lying lateral recumbency  Lying on side, fully-recumbent  Locomotion  Walking, trotting  Feeding  Taking hay into mouth and/or chewing hay and/or grazing and/or browsing  Drinking  Consuming water  Ruminating standing  Standing, generally with a relaxed posture with regular chewing and regurgitation movements  Ruminating lying  As above, but lying sternum  Licking standing/lying  Standing or lying on sternum, turning to lick or attempt to lick body (body region noted)  Rub/scratch  Rubbing/scratching head or body against an object  Vocalization  Bellow or low  Teeth-grinding  Grinding molars together  Shiver/tremble  Whole of body shivering, shaking or trembling  Butt  Butt or attempted butt directed at another animal  Charge  Charges at another animal and stops  Push  Pushes another animal out of the way  Chase  Chases another animal (pursuit continues for some seconds)  Retreat  Moves away from butt, charge, push or chase  Grooms another  Licks another animal  Receives grooming  Recipient of grooming  View Large Table 1. Ethogram developed for investigating changes in the behavior of heifers and cows in response to restraint, mock AI, electroimmobilization, spaying by the dropped ovary technique, and flank spaying with electroimmobilization Behavior  Description  Standing head down  Head level with or below brisket  Standing head up  Head above brisket  Standing stiff-tailed  Standing with tail held stiffly away from body  Lying sternal recumbency  Lying on sternum or partially on sternum with hind-quarters to one side  Lying lateral recumbency  Lying on side, fully-recumbent  Locomotion  Walking, trotting  Feeding  Taking hay into mouth and/or chewing hay and/or grazing and/or browsing  Drinking  Consuming water  Ruminating standing  Standing, generally with a relaxed posture with regular chewing and regurgitation movements  Ruminating lying  As above, but lying sternum  Licking standing/lying  Standing or lying on sternum, turning to lick or attempt to lick body (body region noted)  Rub/scratch  Rubbing/scratching head or body against an object  Vocalization  Bellow or low  Teeth-grinding  Grinding molars together  Shiver/tremble  Whole of body shivering, shaking or trembling  Butt  Butt or attempted butt directed at another animal  Charge  Charges at another animal and stops  Push  Pushes another animal out of the way  Chase  Chases another animal (pursuit continues for some seconds)  Retreat  Moves away from butt, charge, push or chase  Grooms another  Licks another animal  Receives grooming  Recipient of grooming  Behavior  Description  Standing head down  Head level with or below brisket  Standing head up  Head above brisket  Standing stiff-tailed  Standing with tail held stiffly away from body  Lying sternal recumbency  Lying on sternum or partially on sternum with hind-quarters to one side  Lying lateral recumbency  Lying on side, fully-recumbent  Locomotion  Walking, trotting  Feeding  Taking hay into mouth and/or chewing hay and/or grazing and/or browsing  Drinking  Consuming water  Ruminating standing  Standing, generally with a relaxed posture with regular chewing and regurgitation movements  Ruminating lying  As above, but lying sternum  Licking standing/lying  Standing or lying on sternum, turning to lick or attempt to lick body (body region noted)  Rub/scratch  Rubbing/scratching head or body against an object  Vocalization  Bellow or low  Teeth-grinding  Grinding molars together  Shiver/tremble  Whole of body shivering, shaking or trembling  Butt  Butt or attempted butt directed at another animal  Charge  Charges at another animal and stops  Push  Pushes another animal out of the way  Chase  Chases another animal (pursuit continues for some seconds)  Retreat  Moves away from butt, charge, push or chase  Grooms another  Licks another animal  Receives grooming  Recipient of grooming  View Large The observations of the replicate groups on d 0 were conducted when groups were held in yards between blood sampling. Some yards did not allow unimpeded viewing of the cattle, and cattle were also moved between yards as they were moved to the squeeze-chute for sampling. As a consequence, a variable number (range 13 to 30) of observations were made on each replicate group and at differing frequencies, but covering the 7.5-h period posttreatment. Generally, 3 or 4 consecutive scans (Martin and Bateson, 1986) of a group were made at 5-min intervals, and at each scan, the behavior of each individual was recorded according to the ethogram (i.e., the observer would successively determine the identification of individuals in the group and record their behavior at that point in time until all had been recorded, then 5 min later the procedure was repeated). As each replicate group contained 2 animals from each treatment, treatments had equal numbers of recordings. The order in which the animals within groups was recorded was according to the observer being able to read the ear-tag number. For data analyses, the times of day were converted to time after the procedures had been conducted. When the 5 replicate groups were combined in the yard at the end of d 0, the observer scanned the group at 10-min intervals and tallied the numbers of animals, according to ear-tag color, performing the behaviors given in the ethogram. These observations were conducted until it became too dark to distinguish ear-tag colors. For the first group (50 heifers), the observations were between 1635 and 1820 h; for the second (50 cows), 1705 and 1825 h; and for the third (50 heifers), between 1715 and 1825 h. Paddock observations were conducted on the cattle in the afternoon after their 24-h posttreatment blood sample, and in the mornings and afternoons of the next 2 d. Morning observations commenced as soon as it was light enough to discern ear-tag colors (about 0700 h) until about 1100 h. Afternoon observations were between approximately 1500 and 1815 h, except for the third afternoon for each paddock group, because the cattle were mustered at about 1700 h to be taken to the yards in preparation for blood sampling the next morning. Observations were conducted from 4-wheel-drive motorbikes using binoculars, with care taken to move through the paddocks as quietly and slowly as possible to minimize disturbance of the cattle. When cattle were located, the observers stopped their motorbikes at some distance (a minimum of about 20 m), switched off the engines, and waited for 2 to 3 min for the cattle to cease monitoring the observers. Observers then tallied the number of animals, according to ear-tag color, performing the behaviors given in the ethogram. When the cattle were together in a certain part of the paddock, the observations were conducted at 10-min intervals but, frequently, the herd was split into a number of subgroups in different locations in the paddock and it was necessary to drive between groups to conduct the observations. As a consequence, the intervals between the observations were variable (10 to 30 min) and, therefore, the number of observation within each time period (morning and afternoon) and between days varied (6 to 24). Occasionally, not all cattle were located at every scan sample and, therefore, for data analysis, tallies were expressed as percentages of the number of observations. Morbidity, Mortality, and Productivity Recordings The general health status of the cattle was visually assessed until d 4 during blood sampling and behavioral observations. Thereafter, cattle were checked daily for 1 wk postprocedures and then every 2 to 3 d during water supply checks. Cattle that appeared clinically unwell (away from the other cattle, not grazing, walking slowly, or recumbent) were examined from a distance initially and then, in some cases, a closer general physical examination was performed (primarily on recumbent cattle). At 21 and 42 d postprocedures, flank incision wounds were visually assessed and scored using a 5-point scale (1 = wound closed, no inflammation or discharge; 2 = wound partially dehisced, no inflammation or discharge; 3 = wound partially dehisced, no inflammation, but some discharge; 4 = wound completely dehisced and inflamed, but no discharge; and 5 = wound completely dehisced, and inflamed with discharge). Body weight and fat depth were recorded at 4, 21, and 42 d postprocedures, and ADG was calculated based on these BW. Body weights were recorded after a 12-h feed withdrawal period using a Tru-test XR3000 data logger (Tru-test Pty Ltd., Sunnybank, QLD, Australia) connected to HD1010 weigh bars fixed beneath a CIA immobilizer crush/weigh box (Leicht's Country Industries Australia, Goombungee, QLD, Australia). Fat depth at the P8 site (which is located over the gluteal muscles of the rump at the intersection of a perpendicular line through the third sacral vertebra with a line through the tuber ischii and parallel with the vertebral column) was measured using an Ultramac B-10 (Advanced Measurement and Control Pty Ltd., Armidale, NSW, Australia) cattle fat depth meter. Statistical Analyses The statistical design was a randomized complete block design, with the animal as the experimental unit. All analyses were conducted using GenStat for Windows (GenStat, 2008). For the animal behavioral analyses, the counts were pooled into relevant time and treatment categories. These were subjected to generalized linear modeling (McCullagh and Nelder, 1989), using the Binomial distribution with the logit link. Results are presented as proportions or percentages of the total number of observations. Regarding the blood chemistry variables, each animal was successively sampled over time, so a split-plot repeated-measures ANOVA (Rowell and Walters, 1976) was adopted, using the appropriate error term for each factor and interaction. The Greenhouse-Geisser epsilon was estimated to account for the degree of temporal autocorrelation, and the significance levels of the F-tests were appropriately adjusted for this. Residual graphics were used to check for nonnormality of the residuals and heterogeneity of the variances. For the variables where this was found, the log10 transformation was adopted. After this transformation, all residuals were approximately normally distributed with homogeneous variances. To adjust for preexisting animal differences, all variables were measured before the treatments being applied, and these measures were then used as covariates in the respective analyses. Initial BW and flight speeds had been used to achieve a balanced allocation of the animals to blocks, so these variables were not included in the subsequent analyses. Although no statistical outliers were identified, it was noted that 1 animal died from causes unrelated to the treatments (dehorning-related sepsis). Analyses were rerun omitting this animal; however, as the results were virtually unchanged (and did not improve the precision), results from the all-data analyses were retained. Exploratory spline and regression models over time were investigated; however, these tended to smooth out some of the treatment responses which proved to be of specific interest. Hence, the individual treatment by time means (adjusted for the covariate of initial value) from the repeated-measures ANOVA are presented. Analyses of variance of areas under the time-curves were also conducted; however, these gave very similar results to the analyses of the actual (or log-transformed) values. Areas under the curve can only be interpreted relative to a chosen baseline treatment (such as the control). Actual values can also be interpreted this way, but in addition are meaningful in their own right and, as such, were selected for presentation. As 12 out of 42 F-tests for the treatment by class (heifer or cow) and treatment by class by time interactions were significant (P < 0.05), we concluded that cows and heifers did tend to respond differently. Hence, separate means for each class are presented. For the analyses of log10-transformed data, all back-transformed means presented have not used the bias-correction factor (Kendall et al., 1983), so these values are the geometric means. To assist with interpretation, all data variables were re-grouped and analyzed according to 3 consistent time periods, namely 0 to 8, 8 to 24, and 24 to 96 h. Protected significant-difference testing (using LSD at P = 0.05) was used to determine significant differences between the treatment means. RESULTS Morbidity, Mortality, Growth Rate, and Fat Thickness On the day procedures were conducted, immediately after release from the crush, 3 FL cows showed signs of mild to moderate left radial nerve palsy. They were monitored throughout the day and all had significantly improved by 8 h after procedures, although 1 cow still had an abnormal gait which persisted throughout the experiment. A FL heifer that was noted as unwell during behavioral observations died at d 5 postprocedure and necropsy revealed evidence of acute diffuse peritonitis. A PR heifer died at d 12 as a result of dehorning wound-related sepsis. Two PR cows and 1 FL heifer were missing at d 21 and 42, but as no carcasses were found in the study paddocks, it was presumed that they had strayed into an adjoining paddock. There were no significant differences in BW change among treatments; all cattle lost BW between d 0 and 4 [overall averages of –0.98 ± 0.34 kg/d for the cows (P = 0.63) and –0.59 ± 0.16 kg/d for the heifers (P = 0.28)], and during the 42 d of the trial [–0.136 ± 0.047 kg/d for the cows (P = 0.10) and –0.041 ± 0.014 kg/d for the heifers (P = 0.71)]. Additionally, P8 fat depth for the heifers was not different between treatments at d 21 (P = 0.15; overall mean of 1.11 ± 0.04 mm) or d 42 (P = 0.70; 1.24 ± 0.06 mm). For the cows, there was no significant difference between treatments at d 21 (P = 0.17; 14.1 ± 1.4 mm), but at d 42, the FL group had a significantly (P < 0.05) lower fat depth (6.3 mm) than the PR (17.1 mm) and DOT (14.4 mm) treatments, between which there was no significant difference (SE = 2.4). There was also no significant difference between the fat depths of DOT, EIM (9.6 mm), and MAI (7.6 mm) groups. Sixteen percent of FL heifers showed signs of prolonged or abnormal wound healing, with wound scores 3 (partially dehisced with no inflammation, but a slight discharge) and 4 (wound completely dehisced and inflamed, but no discharge) on d 42 (Table 2). At d 42, the flank incisions in the cows had either healed or were partially healed without any evidence of wound infection. Table 2. Frequency of wound healing scores at 21 and 42 d after flank spaying Class  d  n  Wound score1  1  2  3  4  5        %  Heifers  21  18  0  72  22  6  0    42  19  68  16  11  5  0  Cows  21  8  0  63  25  0  13    42  8  88  13  0  0  0  Class  d  n  Wound score1  1  2  3  4  5        %  Heifers  21  18  0  72  22  6  0    42  19  68  16  11  5  0  Cows  21  8  0  63  25  0  13    42  8  88  13  0  0  0  1Wound score: 1 = wound closed, no inflammation or discharge; 2 = wound partially dehisced, no inflammation or discharge; 3 = wound partially dehisced, no inflammation, some discharge; 4 = wound completely dehisced and inflamed, no discharge; 5 = wound completely dehisced, and inflamed with discharge. View Large Table 2. Frequency of wound healing scores at 21 and 42 d after flank spaying Class  d  n  Wound score1  1  2  3  4  5        %  Heifers  21  18  0  72  22  6  0    42  19  68  16  11  5  0  Cows  21  8  0  63  25  0  13    42  8  88  13  0  0  0  Class  d  n  Wound score1  1  2  3  4  5        %  Heifers  21  18  0  72  22  6  0    42  19  68  16  11  5  0  Cows  21  8  0  63  25  0  13    42  8  88  13  0  0  0  1Wound score: 1 = wound closed, no inflammation or discharge; 2 = wound partially dehisced, no inflammation or discharge; 3 = wound partially dehisced, no inflammation, some discharge; 4 = wound completely dehisced and inflamed, no discharge; 5 = wound completely dehisced, and inflamed with discharge. View Large Blood Constituents At most time points during the 0- to 8-h period, bound cortisol concentrations were significantly greater in FL, DOT, and EIM cows compared with PR and MAI (Fig. 1). For heifers, the magnitude of differences was less obvious, but overall mean bound cortisol concentrations for the 0- to 8-h period were significantly greater (P < 0.05) in the FL and DOT compared with PR, with MAI and EIM values intermediate (Table 3). In this same period in the cows, mean cortisol concentrations were significantly greater in FL, DOT, and EIM cows compared with PR and MAI cows. In the 8- to 24-h period, a similar pattern persisted in the cows, with the FL treatment having significantly (P < 0.05) greater mean bound cortisol concentrations compared with all other treatments, and DOT and EIM cows having significantly (P < 0.05) greater concentrations than MAI, but not PR cows. Mean bound cortisol concentrations were similar (P = 0.75) among treatments for the heifers during this period, but in the 24- to 96-h period, significantly greater concentrations were again found in the FL, and also the MAI, compared with the PR heifers. During the 24- to 96-h period, significantly (P < 0.05) greater mean bound cortisol concentrations were found in the FL compared with the MAI and EIM cows. Plasma free cortisol concentrations of heifers did not differ among treatments in any time period postprocedures (P = 0.25 to 0.66), but in cows, concentrations were significantly (P < 0.05) greater in FL, DOT, EIM, and MAI compared with PR during the 0- to 8-h period (Table 3). In the 8- to 24-h period, concentrations were significantly (P < 0.05) greater in DOT and EIM compared with PR cows. Table 3. Mean plasma concentrations (and SE) during 3 time periods postprocedures for heifers and cows subjected to physical restraint (PR), PR and mock AI (MAI), PR and electroimmobilization (EIM), PR and dropped ovary spaying (DOT), or PR, electroimmobilization, and flank spaying (FL)   0 to 8 h  8 to 24 h  24 to 96 h  Constituents1  PR  MAI  EIM  DOT  FL  SE  PR  MAI  EIM  DOT  FL  SE  PR  MAI  EIM  DOT  FL  SE  Heifers (n = 99)      Bound cortisol, nmol/L  2.408a (255.6)  2.450ab (281.5)  2.469ab (294.4)  2.556b (359.7)  2.560b (363.2)  0.042  2.328 (212.8)  2.371 (235.0)  2.368 (233.3)  2.377 (238.2)  2.359 (228.6)  0.049  2.342a (219.8)  2.524b (334.2)  2.448ab (280.5)  2.403ab (252.9)  2.483b (304.1)  0.050      Free cortisol, nmol/L  0.858 (7.21)  0.841 (6.93)  0.884 (7.66)  0.916 (8.24)  0.918 (8.28)  0.039  1.035 (10.84)  1.057 (11.40)  1.105 (12.74)  1.118 (13.12)  1.041 (10.99)  0.038  1.309 (20.37)  1.355 (22.65)  1.344 (22.08)  1.365 (23.16)  1.367 (23.28)  0.033      Haptoglobin, mg/L  Not assayed  0.181a  0.210a  0.214a  0.280a  0.421b  1.049  0.241a  0.386ab  0.258a  0.337a  0.541b  0.061      NEFA, nmol/L  0.650  0.573  0.490  0.610  0.647  0.069  0.651  0.677  0.666  0.677  0.699  0.071  0.831  0.941  1.028  0.916  0.792  0.076      CK,2 IU/L  3.019a (1045)  3.053a (1130)  3.557b (3606)  3.179a (1510)  3.490b (3090)  0.087  3.032a (1077)  2.973a (940)  3.331bc (2142)  3.159ab (1442)  3.475c (2984)  0.087  2.564a (366)  2.490a (309)  2.835bc (684)  2.608ab (406)  3.056c (1138)  0.091      AST,3 IU/L  1.917a (82.5)  1.957a (90.6)  2.189b (154.4)  1.988a (97.3)  2.124b (133.1)  0.029  1.984a (96.3)  1.971a (93.6)  2.126bc (133.7)  2.031ab (107.4)  2.199c (158.1)  0.038  1.939a (86.8)  1.924a (83.9)  2.091b (123.4)  1.913a (81.8)  2.184b (152.7)  0.047  Cows (n = 50)      Bound cortisol, nmol/L  2.198a (157.6)  2.249a (177.5)  2.416b (260.4)  2.472b (296.3)  2.476b (299.4)  0.042  2.267ab (184.9)  2.187a (153.8)  2.370b (234.4)  2.351b (224.4)  2.524c (334.2)  0.049  2.451ab (282.5)  2.330a (213.8)  2.360a (229.1)  2.445ab (278.6)  2.542b (348.3)  0.050      Free cortisol, nmol/L  0.558a (3.61)  0.767b (5.85)  0.855bc (7.16)  0.890c (7.76)  0.820bc (6.61)  0.039  1.050a (11.22)  1.086ab (12.19)  1.161bc (14.49)  1.198c (15.78)  1.116abc (13.06)  0.038  1.388 (24.43)  1.433 (27.10)  1.493 (31.12)  1.433 (27.07)  1.425 (26.61)  0.033      Haptoglobin, mg/L  Not assayed  0.273  0.333  0.401  0.243  0.358  0.049  0.286a  0.445ab  0.367ab  0.522b  0.511b  0.061      NEFA, NMOL/L  0.518abc  0.422a  0.483ab  0.722c  0.641bc  0.069  0.553  0.518  0.616  0.794  0.665  0.071  0.924  0.783  1.038  1.096  0.990  0.076      CK, IU/L  2.687a (486)  2.968b (929)  3.339bc (2183)  3.045b (1109)  3.361c (2296)  0.087  2.666a (463)  2.910b (812)  3.390c (2454)  2.957b (906)  3.313c (2056)  0.087  2.495 (313)  2.607 (405)  3.009 (1021)  2.558 (361)  2.989 (975)  0.091      AST, IU/L  1.857a (71.9)  1.885ab (76.8)  1.959bc (91.1)  1.914abc (82.1)  1.982c (95.9)  0.029  1.869a (74.0)  1.908a (80.8)  2.037b (108.9)  1.939ab (86.8)  2.037b (108.9)  0.038  1.876a (75.1)  1.870a (74.2)  2.057b (113.9)  1.941ab (87.2)  2.191c (155.4)  0.047    0 to 8 h  8 to 24 h  24 to 96 h  Constituents1  PR  MAI  EIM  DOT  FL  SE  PR  MAI  EIM  DOT  FL  SE  PR  MAI  EIM  DOT  FL  SE  Heifers (n = 99)      Bound cortisol, nmol/L  2.408a (255.6)  2.450ab (281.5)  2.469ab (294.4)  2.556b (359.7)  2.560b (363.2)  0.042  2.328 (212.8)  2.371 (235.0)  2.368 (233.3)  2.377 (238.2)  2.359 (228.6)  0.049  2.342a (219.8)  2.524b (334.2)  2.448ab (280.5)  2.403ab (252.9)  2.483b (304.1)  0.050      Free cortisol, nmol/L  0.858 (7.21)  0.841 (6.93)  0.884 (7.66)  0.916 (8.24)  0.918 (8.28)  0.039  1.035 (10.84)  1.057 (11.40)  1.105 (12.74)  1.118 (13.12)  1.041 (10.99)  0.038  1.309 (20.37)  1.355 (22.65)  1.344 (22.08)  1.365 (23.16)  1.367 (23.28)  0.033      Haptoglobin, mg/L  Not assayed  0.181a  0.210a  0.214a  0.280a  0.421b  1.049  0.241a  0.386ab  0.258a  0.337a  0.541b  0.061      NEFA, nmol/L  0.650  0.573  0.490  0.610  0.647  0.069  0.651  0.677  0.666  0.677  0.699  0.071  0.831  0.941  1.028  0.916  0.792  0.076      CK,2 IU/L  3.019a (1045)  3.053a (1130)  3.557b (3606)  3.179a (1510)  3.490b (3090)  0.087  3.032a (1077)  2.973a (940)  3.331bc (2142)  3.159ab (1442)  3.475c (2984)  0.087  2.564a (366)  2.490a (309)  2.835bc (684)  2.608ab (406)  3.056c (1138)  0.091      AST,3 IU/L  1.917a (82.5)  1.957a (90.6)  2.189b (154.4)  1.988a (97.3)  2.124b (133.1)  0.029  1.984a (96.3)  1.971a (93.6)  2.126bc (133.7)  2.031ab (107.4)  2.199c (158.1)  0.038  1.939a (86.8)  1.924a (83.9)  2.091b (123.4)  1.913a (81.8)  2.184b (152.7)  0.047  Cows (n = 50)      Bound cortisol, nmol/L  2.198a (157.6)  2.249a (177.5)  2.416b (260.4)  2.472b (296.3)  2.476b (299.4)  0.042  2.267ab (184.9)  2.187a (153.8)  2.370b (234.4)  2.351b (224.4)  2.524c (334.2)  0.049  2.451ab (282.5)  2.330a (213.8)  2.360a (229.1)  2.445ab (278.6)  2.542b (348.3)  0.050      Free cortisol, nmol/L  0.558a (3.61)  0.767b (5.85)  0.855bc (7.16)  0.890c (7.76)  0.820bc (6.61)  0.039  1.050a (11.22)  1.086ab (12.19)  1.161bc (14.49)  1.198c (15.78)  1.116abc (13.06)  0.038  1.388 (24.43)  1.433 (27.10)  1.493 (31.12)  1.433 (27.07)  1.425 (26.61)  0.033      Haptoglobin, mg/L  Not assayed  0.273  0.333  0.401  0.243  0.358  0.049  0.286a  0.445ab  0.367ab  0.522b  0.511b  0.061      NEFA, NMOL/L  0.518abc  0.422a  0.483ab  0.722c  0.641bc  0.069  0.553  0.518  0.616  0.794  0.665  0.071  0.924  0.783  1.038  1.096  0.990  0.076      CK, IU/L  2.687a (486)  2.968b (929)  3.339bc (2183)  3.045b (1109)  3.361c (2296)  0.087  2.666a (463)  2.910b (812)  3.390c (2454)  2.957b (906)  3.313c (2056)  0.087  2.495 (313)  2.607 (405)  3.009 (1021)  2.558 (361)  2.989 (975)  0.091      AST, IU/L  1.857a (71.9)  1.885ab (76.8)  1.959bc (91.1)  1.914abc (82.1)  1.982c (95.9)  0.029  1.869a (74.0)  1.908a (80.8)  2.037b (108.9)  1.939ab (86.8)  2.037b (108.9)  0.038  1.876a (75.1)  1.870a (74.2)  2.057b (113.9)  1.941ab (87.2)  2.191c (155.4)  0.047  a–cWithin a row, means without a common letter differ (P < 0.05). 1Analyzed and presented on log scale, except for Haptoglobin and NEFA, with means on the measured scale in parentheses. 2CK = creatine kinase. 3AST = aspartate aminotransferase. View Large Table 3. Mean plasma concentrations (and SE) during 3 time periods postprocedures for heifers and cows subjected to physical restraint (PR), PR and mock AI (MAI), PR and electroimmobilization (EIM), PR and dropped ovary spaying (DOT), or PR, electroimmobilization, and flank spaying (FL)   0 to 8 h  8 to 24 h  24 to 96 h  Constituents1  PR  MAI  EIM  DOT  FL  SE  PR  MAI  EIM  DOT  FL  SE  PR  MAI  EIM  DOT  FL  SE  Heifers (n = 99)      Bound cortisol, nmol/L  2.408a (255.6)  2.450ab (281.5)  2.469ab (294.4)  2.556b (359.7)  2.560b (363.2)  0.042  2.328 (212.8)  2.371 (235.0)  2.368 (233.3)  2.377 (238.2)  2.359 (228.6)  0.049  2.342a (219.8)  2.524b (334.2)  2.448ab (280.5)  2.403ab (252.9)  2.483b (304.1)  0.050      Free cortisol, nmol/L  0.858 (7.21)  0.841 (6.93)  0.884 (7.66)  0.916 (8.24)  0.918 (8.28)  0.039  1.035 (10.84)  1.057 (11.40)  1.105 (12.74)  1.118 (13.12)  1.041 (10.99)  0.038  1.309 (20.37)  1.355 (22.65)  1.344 (22.08)  1.365 (23.16)  1.367 (23.28)  0.033      Haptoglobin, mg/L  Not assayed  0.181a  0.210a  0.214a  0.280a  0.421b  1.049  0.241a  0.386ab  0.258a  0.337a  0.541b  0.061      NEFA, nmol/L  0.650  0.573  0.490  0.610  0.647  0.069  0.651  0.677  0.666  0.677  0.699  0.071  0.831  0.941  1.028  0.916  0.792  0.076      CK,2 IU/L  3.019a (1045)  3.053a (1130)  3.557b (3606)  3.179a (1510)  3.490b (3090)  0.087  3.032a (1077)  2.973a (940)  3.331bc (2142)  3.159ab (1442)  3.475c (2984)  0.087  2.564a (366)  2.490a (309)  2.835bc (684)  2.608ab (406)  3.056c (1138)  0.091      AST,3 IU/L  1.917a (82.5)  1.957a (90.6)  2.189b (154.4)  1.988a (97.3)  2.124b (133.1)  0.029  1.984a (96.3)  1.971a (93.6)  2.126bc (133.7)  2.031ab (107.4)  2.199c (158.1)  0.038  1.939a (86.8)  1.924a (83.9)  2.091b (123.4)  1.913a (81.8)  2.184b (152.7)  0.047  Cows (n = 50)      Bound cortisol, nmol/L  2.198a (157.6)  2.249a (177.5)  2.416b (260.4)  2.472b (296.3)  2.476b (299.4)  0.042  2.267ab (184.9)  2.187a (153.8)  2.370b (234.4)  2.351b (224.4)  2.524c (334.2)  0.049  2.451ab (282.5)  2.330a (213.8)  2.360a (229.1)  2.445ab (278.6)  2.542b (348.3)  0.050      Free cortisol, nmol/L  0.558a (3.61)  0.767b (5.85)  0.855bc (7.16)  0.890c (7.76)  0.820bc (6.61)  0.039  1.050a (11.22)  1.086ab (12.19)  1.161bc (14.49)  1.198c (15.78)  1.116abc (13.06)  0.038  1.388 (24.43)  1.433 (27.10)  1.493 (31.12)  1.433 (27.07)  1.425 (26.61)  0.033      Haptoglobin, mg/L  Not assayed  0.273  0.333  0.401  0.243  0.358  0.049  0.286a  0.445ab  0.367ab  0.522b  0.511b  0.061      NEFA, NMOL/L  0.518abc  0.422a  0.483ab  0.722c  0.641bc  0.069  0.553  0.518  0.616  0.794  0.665  0.071  0.924  0.783  1.038  1.096  0.990  0.076      CK, IU/L  2.687a (486)  2.968b (929)  3.339bc (2183)  3.045b (1109)  3.361c (2296)  0.087  2.666a (463)  2.910b (812)  3.390c (2454)  2.957b (906)  3.313c (2056)  0.087  2.495 (313)  2.607 (405)  3.009 (1021)  2.558 (361)  2.989 (975)  0.091      AST, IU/L  1.857a (71.9)  1.885ab (76.8)  1.959bc (91.1)  1.914abc (82.1)  1.982c (95.9)  0.029  1.869a (74.0)  1.908a (80.8)  2.037b (108.9)  1.939ab (86.8)  2.037b (108.9)  0.038  1.876a (75.1)  1.870a (74.2)  2.057b (113.9)  1.941ab (87.2)  2.191c (155.4)  0.047    0 to 8 h  8 to 24 h  24 to 96 h  Constituents1  PR  MAI  EIM  DOT  FL  SE  PR  MAI  EIM  DOT  FL  SE  PR  MAI  EIM  DOT  FL  SE  Heifers (n = 99)      Bound cortisol, nmol/L  2.408a (255.6)  2.450ab (281.5)  2.469ab (294.4)  2.556b (359.7)  2.560b (363.2)  0.042  2.328 (212.8)  2.371 (235.0)  2.368 (233.3)  2.377 (238.2)  2.359 (228.6)  0.049  2.342a (219.8)  2.524b (334.2)  2.448ab (280.5)  2.403ab (252.9)  2.483b (304.1)  0.050      Free cortisol, nmol/L  0.858 (7.21)  0.841 (6.93)  0.884 (7.66)  0.916 (8.24)  0.918 (8.28)  0.039  1.035 (10.84)  1.057 (11.40)  1.105 (12.74)  1.118 (13.12)  1.041 (10.99)  0.038  1.309 (20.37)  1.355 (22.65)  1.344 (22.08)  1.365 (23.16)  1.367 (23.28)  0.033      Haptoglobin, mg/L  Not assayed  0.181a  0.210a  0.214a  0.280a  0.421b  1.049  0.241a  0.386ab  0.258a  0.337a  0.541b  0.061      NEFA, nmol/L  0.650  0.573  0.490  0.610  0.647  0.069  0.651  0.677  0.666  0.677  0.699  0.071  0.831  0.941  1.028  0.916  0.792  0.076      CK,2 IU/L  3.019a (1045)  3.053a (1130)  3.557b (3606)  3.179a (1510)  3.490b (3090)  0.087  3.032a (1077)  2.973a (940)  3.331bc (2142)  3.159ab (1442)  3.475c (2984)  0.087  2.564a (366)  2.490a (309)  2.835bc (684)  2.608ab (406)  3.056c (1138)  0.091      AST,3 IU/L  1.917a (82.5)  1.957a (90.6)  2.189b (154.4)  1.988a (97.3)  2.124b (133.1)  0.029  1.984a (96.3)  1.971a (93.6)  2.126bc (133.7)  2.031ab (107.4)  2.199c (158.1)  0.038  1.939a (86.8)  1.924a (83.9)  2.091b (123.4)  1.913a (81.8)  2.184b (152.7)  0.047  Cows (n = 50)      Bound cortisol, nmol/L  2.198a (157.6)  2.249a (177.5)  2.416b (260.4)  2.472b (296.3)  2.476b (299.4)  0.042  2.267ab (184.9)  2.187a (153.8)  2.370b (234.4)  2.351b (224.4)  2.524c (334.2)  0.049  2.451ab (282.5)  2.330a (213.8)  2.360a (229.1)  2.445ab (278.6)  2.542b (348.3)  0.050      Free cortisol, nmol/L  0.558a (3.61)  0.767b (5.85)  0.855bc (7.16)  0.890c (7.76)  0.820bc (6.61)  0.039  1.050a (11.22)  1.086ab (12.19)  1.161bc (14.49)  1.198c (15.78)  1.116abc (13.06)  0.038  1.388 (24.43)  1.433 (27.10)  1.493 (31.12)  1.433 (27.07)  1.425 (26.61)  0.033      Haptoglobin, mg/L  Not assayed  0.273  0.333  0.401  0.243  0.358  0.049  0.286a  0.445ab  0.367ab  0.522b  0.511b  0.061      NEFA, NMOL/L  0.518abc  0.422a  0.483ab  0.722c  0.641bc  0.069  0.553  0.518  0.616  0.794  0.665  0.071  0.924  0.783  1.038  1.096  0.990  0.076      CK, IU/L  2.687a (486)  2.968b (929)  3.339bc (2183)  3.045b (1109)  3.361c (2296)  0.087  2.666a (463)  2.910b (812)  3.390c (2454)  2.957b (906)  3.313c (2056)  0.087  2.495 (313)  2.607 (405)  3.009 (1021)  2.558 (361)  2.989 (975)  0.091      AST, IU/L  1.857a (71.9)  1.885ab (76.8)  1.959bc (91.1)  1.914abc (82.1)  1.982c (95.9)  0.029  1.869a (74.0)  1.908a (80.8)  2.037b (108.9)  1.939ab (86.8)  2.037b (108.9)  0.038  1.876a (75.1)  1.870a (74.2)  2.057b (113.9)  1.941ab (87.2)  2.191c (155.4)  0.047  a–cWithin a row, means without a common letter differ (P < 0.05). 1Analyzed and presented on log scale, except for Haptoglobin and NEFA, with means on the measured scale in parentheses. 2CK = creatine kinase. 3AST = aspartate aminotransferase. View Large Figure 1. View largeDownload slide Mean changes in log bound cortisol concentrations for (a) heifers and (b) cows in the 8 h after procedures were performed: physical restraint (PR); PR and mock AI (MAI); PR and electroimmobilization (EIM); PR and spaying by the dropped ovary technique (DOT); and PR, electroimmobilization, and flank spaying (FL). Bars are the SE of the mean of the PR treatment. Figure 1. View largeDownload slide Mean changes in log bound cortisol concentrations for (a) heifers and (b) cows in the 8 h after procedures were performed: physical restraint (PR); PR and mock AI (MAI); PR and electroimmobilization (EIM); PR and spaying by the dropped ovary technique (DOT); and PR, electroimmobilization, and flank spaying (FL). Bars are the SE of the mean of the PR treatment. Serum haptoglobin concentrations were not assayed in samples taken during the 0- to 8-h period. In the 8- to 24-h period, haptoglobin concentrations were similar (P = 0.68) among treatments in cows, but were significantly (P < 0.05) greater in FL heifers compared with all other treatments (Table 3). FL heifers also had significantly (P < 0.05) greater haptoglobin concentrations compared with PR, DOT, and EIM heifers in the 24- to 96-h period. In contrast, both FL and DOT cows had significantly (P < 0.05) greater haptoglobin concentrations compared with PR cows in this same period. Plasma NEFA concentrations were not affected (P = 0.48 to 0.61) by treatment in any time period in the heifers. However, FL and DOT cows had significantly (P < 0.05) greater concentrations then MAI cows during the 0- to 8-h period (Table 3). During the 0- to 8-h period, FL and EIM heifers had significantly (P < 0.05) greater concentrations of CK and AST compared with PR, MAI, and DOT (Table 3). Additionally, plasma CK and AST concentrations were greater (P < 0.05) in FL than PR, MAI, and DOT heifers in both the 8- to 24-h and 24- to 96-h periods. In the cows, the pattern was similar: in the 0- to 8-h period, FL cows had greater (P < 0.05) concentrations of CK compared with PR, MAI, and DOT cows, but AST concentrations were only greater (P < 0.05) compared with PR and MAI. In the 8- to 24-h period, both CK and AST concentrations were greater (P < 0.05) in FL cows compared with PR and MAI and, additionally, CK concentrations were similar between FL and EIM cows. In the 24 to 96-h period, AST was greater (P < 0.05) in the FL cows compared with all other treatments. Behavior As some behaviors were recorded infrequently, they could not be analyzed. Only behaviors with sufficient numbers of recordings for statistical analysis are presented in Table 4. During d 0, significantly (P < 0.05) more FL heifers and cows stood head down compared with all other treatments. The reciprocal of this pattern was shown for standing head up in the heifers, but this was not observed in the cows. There were insufficient recordings of drinking, ruminating, and standing self-licking, and treatments did not differ for locomotion and lying in sternal recumbency for either heifers or cows during this period. Table 4. Mean percentage of cattle (and SE) performing behaviors during 3 time periods postprocedures when subjected to physical restraint (PR), PR and mock AI (MAI), PR and electroimmobilization (EIM), PR and dropped ovary spaying (DOT) or PR, electroimmobilization and flank spaying (FL)   d 0 (replicate groups observed between blood sampling)  End of d 0 (replicate groups co-mingled and observed in yard)  d 1 to 3 (co-mingled replicate groups observed in paddock)  Behaviors  PR  MAI  EIM  DOT  FL  SE  PR  MAI  EIM  DOT  FL  SE  PR  MAI  EIM  DOT  FL  SE  Heifers (n = 99)      Feeding  food not provided  78.65c  70.22c  73.60c  56.74b  40.11a  3.72  64.82  65.90  69.66  63.44  61.10  2.78      Locomotion  8.33  11.39  10.37  5.61  6.77  2.06  9.55  7.30  7.87  3.93  8.47  2.16  8.91  7.11  6.46  8.48  7.47  1.42      Standing head up  74.98c  65.90b  72.26bc  68.21bc  55.41a  3.19  9.55a  16.29ab  15.73ab  24.16b  35.59c  3.48  8.97  8.94  6.98  9.68  9.97  1.28      Standing head down  3.59a  8.96ab  7.77ab  9.76b  26.26c  2.13  0.56a  1.12a  1.12a  6.18b  5.08b  0.91  0.065a  0.086ab  0.213b  0.216b  0.639c  0.047      Lying sternum  7.47  6.25  2.43  10.45  7.97  1.91  0.00a  1.69b  0.00a  2.81bc  3.39c  0.48  10.92  11.43  11.30  12.66  14.99  1.30      Drinking  insufficient data for analysis  1.12a  0.56a  1.12a  1.12a  3.95b  0.66  0.457  0.486  0.612  0.393  0.449  0.091      Ruminating  insufficient data for analysis  insufficient data for analysis  4.82b  4.55b  3.62ab  4.16ab  2.76A  0.139      Standing self-licking  insufficient data for analysis  insufficient data for analysis  0.653ab  1.016b  0.604a  0.358a  1.791c  0.139  Cows (n = 50)      Feeding  food not provided  56.41b  55.13b  26.92a  30.77a  22.67a  5.68  19.16  24.73  18.04  21.24  21.79  3.05      Locomotion  6.38  11.79  16.56  13.38  6.85  3.39  5.13  16.67  15.38  16.67  12.00  4.16  24.11  22.13  20.16  21.14  20.10  2.77      Standing head up  71.43  70.45  70.60  62.26  65.31  4.70  29.49ab  24.36a  52.56b  43.59b  50.67b  4.70  24.47  27.10  31.26  29.25  26.77  2.54      Standing head down  2.77a  6.82a  1.65a  1.56a  16.47b  2.02  1.28a  0.00a  0.00a  1.28a  9.33b  0.96  0.093a  0.203a  0.455b  0.194a  0.803c  0.072      Lying sternum  5.89  1.76  0.54  1.49  1.84  1.52  5.13b  0.00a  0.00a  1.28a  0.00a  0.51  23.86  17.94  24.11  21.77  22.45  2.25      Drinking  insufficient data for analysis  2.56ab  0.00a  1.28ab  1.28ab  2.67b  0.93  0.000  0.000  0.000  0.000  0.000  0.000      Ruminating  insufficient data for analysis  insufficient data for analysis  7.08  6.91  5.72  5.85  7.04  0.91      Standing self-licking  insufficient data for analysis  insufficient data for analysis  0.529bc  0.349b  0.000a  0.259ab  0.799c  0.120    d 0 (replicate groups observed between blood sampling)  End of d 0 (replicate groups co-mingled and observed in yard)  d 1 to 3 (co-mingled replicate groups observed in paddock)  Behaviors  PR  MAI  EIM  DOT  FL  SE  PR  MAI  EIM  DOT  FL  SE  PR  MAI  EIM  DOT  FL  SE  Heifers (n = 99)      Feeding  food not provided  78.65c  70.22c  73.60c  56.74b  40.11a  3.72  64.82  65.90  69.66  63.44  61.10  2.78      Locomotion  8.33  11.39  10.37  5.61  6.77  2.06  9.55  7.30  7.87  3.93  8.47  2.16  8.91  7.11  6.46  8.48  7.47  1.42      Standing head up  74.98c  65.90b  72.26bc  68.21bc  55.41a  3.19  9.55a  16.29ab  15.73ab  24.16b  35.59c  3.48  8.97  8.94  6.98  9.68  9.97  1.28      Standing head down  3.59a  8.96ab  7.77ab  9.76b  26.26c  2.13  0.56a  1.12a  1.12a  6.18b  5.08b  0.91  0.065a  0.086ab  0.213b  0.216b  0.639c  0.047      Lying sternum  7.47  6.25  2.43  10.45  7.97  1.91  0.00a  1.69b  0.00a  2.81bc  3.39c  0.48  10.92  11.43  11.30  12.66  14.99  1.30      Drinking  insufficient data for analysis  1.12a  0.56a  1.12a  1.12a  3.95b  0.66  0.457  0.486  0.612  0.393  0.449  0.091      Ruminating  insufficient data for analysis  insufficient data for analysis  4.82b  4.55b  3.62ab  4.16ab  2.76A  0.139      Standing self-licking  insufficient data for analysis  insufficient data for analysis  0.653ab  1.016b  0.604a  0.358a  1.791c  0.139  Cows (n = 50)      Feeding  food not provided  56.41b  55.13b  26.92a  30.77a  22.67a  5.68  19.16  24.73  18.04  21.24  21.79  3.05      Locomotion  6.38  11.79  16.56  13.38  6.85  3.39  5.13  16.67  15.38  16.67  12.00  4.16  24.11  22.13  20.16  21.14  20.10  2.77      Standing head up  71.43  70.45  70.60  62.26  65.31  4.70  29.49ab  24.36a  52.56b  43.59b  50.67b  4.70  24.47  27.10  31.26  29.25  26.77  2.54      Standing head down  2.77a  6.82a  1.65a  1.56a  16.47b  2.02  1.28a  0.00a  0.00a  1.28a  9.33b  0.96  0.093a  0.203a  0.455b  0.194a  0.803c  0.072      Lying sternum  5.89  1.76  0.54  1.49  1.84  1.52  5.13b  0.00a  0.00a  1.28a  0.00a  0.51  23.86  17.94  24.11  21.77  22.45  2.25      Drinking  insufficient data for analysis  2.56ab  0.00a  1.28ab  1.28ab  2.67b  0.93  0.000  0.000  0.000  0.000  0.000  0.000      Ruminating  insufficient data for analysis  insufficient data for analysis  7.08  6.91  5.72  5.85  7.04  0.91      Standing self-licking  insufficient data for analysis  insufficient data for analysis  0.529bc  0.349b  0.000a  0.259ab  0.799c  0.120  a–cWithin a row, means without a common letter differ (P < 0.05). View Large Table 4. Mean percentage of cattle (and SE) performing behaviors during 3 time periods postprocedures when subjected to physical restraint (PR), PR and mock AI (MAI), PR and electroimmobilization (EIM), PR and dropped ovary spaying (DOT) or PR, electroimmobilization and flank spaying (FL)   d 0 (replicate groups observed between blood sampling)  End of d 0 (replicate groups co-mingled and observed in yard)  d 1 to 3 (co-mingled replicate groups observed in paddock)  Behaviors  PR  MAI  EIM  DOT  FL  SE  PR  MAI  EIM  DOT  FL  SE  PR  MAI  EIM  DOT  FL  SE  Heifers (n = 99)      Feeding  food not provided  78.65c  70.22c  73.60c  56.74b  40.11a  3.72  64.82  65.90  69.66  63.44  61.10  2.78      Locomotion  8.33  11.39  10.37  5.61  6.77  2.06  9.55  7.30  7.87  3.93  8.47  2.16  8.91  7.11  6.46  8.48  7.47  1.42      Standing head up  74.98c  65.90b  72.26bc  68.21bc  55.41a  3.19  9.55a  16.29ab  15.73ab  24.16b  35.59c  3.48  8.97  8.94  6.98  9.68  9.97  1.28      Standing head down  3.59a  8.96ab  7.77ab  9.76b  26.26c  2.13  0.56a  1.12a  1.12a  6.18b  5.08b  0.91  0.065a  0.086ab  0.213b  0.216b  0.639c  0.047      Lying sternum  7.47  6.25  2.43  10.45  7.97  1.91  0.00a  1.69b  0.00a  2.81bc  3.39c  0.48  10.92  11.43  11.30  12.66  14.99  1.30      Drinking  insufficient data for analysis  1.12a  0.56a  1.12a  1.12a  3.95b  0.66  0.457  0.486  0.612  0.393  0.449  0.091      Ruminating  insufficient data for analysis  insufficient data for analysis  4.82b  4.55b  3.62ab  4.16ab  2.76A  0.139      Standing self-licking  insufficient data for analysis  insufficient data for analysis  0.653ab  1.016b  0.604a  0.358a  1.791c  0.139  Cows (n = 50)      Feeding  food not provided  56.41b  55.13b  26.92a  30.77a  22.67a  5.68  19.16  24.73  18.04  21.24  21.79  3.05      Locomotion  6.38  11.79  16.56  13.38  6.85  3.39  5.13  16.67  15.38  16.67  12.00  4.16  24.11  22.13  20.16  21.14  20.10  2.77      Standing head up  71.43  70.45  70.60  62.26  65.31  4.70  29.49ab  24.36a  52.56b  43.59b  50.67b  4.70  24.47  27.10  31.26  29.25  26.77  2.54      Standing head down  2.77a  6.82a  1.65a  1.56a  16.47b  2.02  1.28a  0.00a  0.00a  1.28a  9.33b  0.96  0.093a  0.203a  0.455b  0.194a  0.803c  0.072      Lying sternum  5.89  1.76  0.54  1.49  1.84  1.52  5.13b  0.00a  0.00a  1.28a  0.00a  0.51  23.86  17.94  24.11  21.77  22.45  2.25      Drinking  insufficient data for analysis  2.56ab  0.00a  1.28ab  1.28ab  2.67b  0.93  0.000  0.000  0.000  0.000  0.000  0.000      Ruminating  insufficient data for analysis  insufficient data for analysis  7.08  6.91  5.72  5.85  7.04  0.91      Standing self-licking  insufficient data for analysis  insufficient data for analysis  0.529bc  0.349b  0.000a  0.259ab  0.799c  0.120    d 0 (replicate groups observed between blood sampling)  End of d 0 (replicate groups co-mingled and observed in yard)  d 1 to 3 (co-mingled replicate groups observed in paddock)  Behaviors  PR  MAI  EIM  DOT  FL  SE  PR  MAI  EIM  DOT  FL  SE  PR  MAI  EIM  DOT  FL  SE  Heifers (n = 99)      Feeding  food not provided  78.65c  70.22c  73.60c  56.74b  40.11a  3.72  64.82  65.90  69.66  63.44  61.10  2.78      Locomotion  8.33  11.39  10.37  5.61  6.77  2.06  9.55  7.30  7.87  3.93  8.47  2.16  8.91  7.11  6.46  8.48  7.47  1.42      Standing head up  74.98c  65.90b  72.26bc  68.21bc  55.41a  3.19  9.55a  16.29ab  15.73ab  24.16b  35.59c  3.48  8.97  8.94  6.98  9.68  9.97  1.28      Standing head down  3.59a  8.96ab  7.77ab  9.76b  26.26c  2.13  0.56a  1.12a  1.12a  6.18b  5.08b  0.91  0.065a  0.086ab  0.213b  0.216b  0.639c  0.047      Lying sternum  7.47  6.25  2.43  10.45  7.97  1.91  0.00a  1.69b  0.00a  2.81bc  3.39c  0.48  10.92  11.43  11.30  12.66  14.99  1.30      Drinking  insufficient data for analysis  1.12a  0.56a  1.12a  1.12a  3.95b  0.66  0.457  0.486  0.612  0.393  0.449  0.091      Ruminating  insufficient data for analysis  insufficient data for analysis  4.82b  4.55b  3.62ab  4.16ab  2.76A  0.139      Standing self-licking  insufficient data for analysis  insufficient data for analysis  0.653ab  1.016b  0.604a  0.358a  1.791c  0.139  Cows (n = 50)      Feeding  food not provided  56.41b  55.13b  26.92a  30.77a  22.67a  5.68  19.16  24.73  18.04  21.24  21.79  3.05      Locomotion  6.38  11.79  16.56  13.38  6.85  3.39  5.13  16.67  15.38  16.67  12.00  4.16  24.11  22.13  20.16  21.14  20.10  2.77      Standing head up  71.43  70.45  70.60  62.26  65.31  4.70  29.49ab  24.36a  52.56b  43.59b  50.67b  4.70  24.47  27.10  31.26  29.25  26.77  2.54      Standing head down  2.77a  6.82a  1.65a  1.56a  16.47b  2.02  1.28a  0.00a  0.00a  1.28a  9.33b  0.96  0.093a  0.203a  0.455b  0.194a  0.803c  0.072      Lying sternum  5.89  1.76  0.54  1.49  1.84  1.52  5.13b  0.00a  0.00a  1.28a  0.00a  0.51  23.86  17.94  24.11  21.77  22.45  2.25      Drinking  insufficient data for analysis  2.56ab  0.00a  1.28ab  1.28ab  2.67b  0.93  0.000  0.000  0.000  0.000  0.000  0.000      Ruminating  insufficient data for analysis  insufficient data for analysis  7.08  6.91  5.72  5.85  7.04  0.91      Standing self-licking  insufficient data for analysis  insufficient data for analysis  0.529bc  0.349b  0.000a  0.259ab  0.799c  0.120  a–cWithin a row, means without a common letter differ (P < 0.05). View Large At the end of d 0 when the replicate groups were co-mingled again, significantly (P < 0.05) more FL cows and heifers stood head down compared with the other treatments and, additionally, more (P < 0.05) DOT heifers stood head down compared with PR, MAI, and EIM heifers. More (P < 0.05) FL and DOT heifers stood head up compared with PR heifers and more (P < 0.05) FL, DOT, and EIM cows stood head up compared with MAI cows. A greater (P < 0.05) percentage of FL and DOT heifers were recorded in sternal recumbency compared with PR and EIM heifers, and the greatest (P < 0.05) percentage of cows in sternal recumbency was in the PR treatment. Fewer (P < 0.05) FL and DOT heifers were observed feeding compared with PR, MAI, and EMI heifers. In addition, fewer (P < 0.05) FL, DOT, and EIM cows fed compared with PR and MAI cows. More (P < 0.05) FL heifers were recorded drinking compared with all other treatments, but more (P < 0.05) FL cows drank compared only with MAI cows. Treatment did not influence the percentage of heifers or cows recorded in locomotion. During paddock observations, more (P < 0.05) FL heifers and FL cows stood head down compared with all other treatments. In addition, more (P < 0.05) DOT and EIM heifers stood head down compared with PR, and more (P < 0.05) EIM cows compared with PR and MAI cows. A decreased (P < 0.05) percentage of FL heifers were recorded ruminating compared with PR and MAI heifers, but there was no effect of treatment on ruminating in the cows. A greater (P < 0.05) percentage of both FL heifers and FL cows were recorded standing self-licking compared with other treatments, although there was no difference between FL and PR cows. Other behaviors were not affected by treatment. DISCUSSION Adverse welfare outcomes relating to morbidity and mortality occurred only in the FL spayed cattle: 1 heifer died with acute diffuse peritonitis as a result of accidental perforation of the intestines during the procedure; 3 cows developed left radial nerve palsy, probably as a consequence of compression of the radial nerve against the head-gate during electroimmobilization; and 16% of heifers showed signs of prolonged or abnormal wound healing. These findings alone indicate that, of the treatments, FL spaying had the greatest adverse effects on cattle welfare. There was consistency between the other welfare indicators used (particularly plasma cortisol concentrations and behavior) that showed that the acute pain (0 to 8 h postprocedures) associated with spaying was not different between DOT and FL techniques in both heifers and cows. It appears, however, from the treatment effects on free cortisol and NEFA, that the pain and stress were more severe and sustained in the cows than the heifers. The increased concentrations of haptoglobin in the FL spayed animals also indicate longer lasting adverse effects, from inflammation, on the welfare of both heifers and cows after FL compared with DOT spaying. We are unable to compare our findings with others because, as is evident from the review by Pinner (2006), the pain and stress associated with spaying has not been previously evaluated. The increased concentrations of CK and AST found in all treatment groups in comparison with the control group indicate that cattle in these groups experienced increased muscular exertion and muscle damage. This was unsurprising for the FL and DOT spayed cattle because the procedures involved the cutting and tearing of smooth and skeletal muscle tissue. The MAI treatment appears to have induced some minor tissue damage in the cows, probably from the rectal palpation and insertion of the AI gun. However, significantly greater concentrations of CK and AST were found in both EIM heifers and cows compared with the controls, probably associated with rupture of fibers and interference with blood supply associated with severe, sustained muscular contraction (Molony, 1986). Stress also seems to be involved in inducing increased CK concentrations via increased muscle membrane permeability, as has been postulated in stress-susceptible pigs (Addis et al., 1974), and increased CK in cattle has been found to be associated with adrenalin injection (McVeigh et al., 1982). Given that increased cortisol concentrations are indicative of stress and pain (Mellor et al., 2000), our findings further indicate that stress and pain are associated with electroimmobilization; plasma concentrations of cortisol in the EIM cattle were generally intermediate between the control and spayed animals, but in the cows there were significantly increased concentrations of bound cortisol in the 0- to 8-h period, and of free cortisol to 24-h postprocedures. This suggestion that electroimmobilization is a noxious experience is further supported by the finding that sheep rapidly developed an aversion to a place where they had been electroimmobilized (Rushen, 1986). The likelihood that electroimmobilization causes stress and pain raises the question of its contribution to the stress and pain of the cattle that were FL spayed. The concentrations of bound cortisol tended to be numerically greatest in the FL and DOT cattle and the cows in particular, whereas CK and AST were, in many instances, similar in the EIM and FL groups. It would seem, therefore, that electroimmobilization made some contribution to the stress and pain, but spaying is painful and stressful regardless of the restraint method used. Our results also indicate that the procedures associated with AI elicited a minor pain or stress response, as evidenced by increased, although generally not significantly different, concentrations in comparison with the PR group, of free cortisol and CK in the MAI cows, and haptoglobin in both MAI heifers and cows. This response to the AI procedure was, however, much less than that evoked by the other procedures. One study on AI in dairy cows reported a peak in plasma cortisol at 10 min, which decreased by 25 to 30 min after the start of the procedure (Nakao et al., 1994). Our blood sampling schedule meant that we did not detect any peak cortisol response, but concentrations were no different between the MAI and the PR groups at 1 h postprocedure. There were some discrepancies between our behavioral and physiological data, in that behavioral responses were similar between cows and heifers, with some behaviors indicating that FL spaying was more painful than DOT at every observation time. The main behaviors that were influenced by treatment were: standing head down for all of the observation periods; feeding, lying and drinking in the observation period at the end of d 0; and self-licking, and ruminating (for heifers only) on the subsequent days when the cattle were in the paddocks. Behavioral responses can be difficult to interpret and, as highlighted by Pinner (2006), research that has systematically examined the behavioral responses of cows or heifers to spaying, or even to manipulation of the reproductive tract as occurs during AI and pregnancy diagnosis, is sparse. This is an important gap in the literature because it would appear that different noxious treatments can elicit unique behavioral responses (Mellor et al., 2000). Recent studies on dairy cows used a subjective “avoidance reactivity score” to assess the behavioral responses of cows to manual vaginal examination (Pilz et al., 2012). The studies showed that, during examination, cows showed an arched back and stretched neck, indicative of discomfort, and the authors suggest that manual vaginal examination is a stressful experience for cows. Our cortisol, CK, AST, and haptoglobin data would indicate that any stress was minor compared with that induced by restraint alone. It must be remembered, however, that dairy cows are more accustomed to being handled than rangeland beef cattle, and are typically not subjected to the degree of restraint to which the cattle in the current study were subjected. Mellor et al. (2000) list vocalizations, temperament, postural, and locomotory changes in response to noxious stimuli and the descriptions included “standing drooping,” “depressed,” and “miserable.” Based on our own experiences of monitoring cattle, these descriptions fit the behavior of cattle that are clinically ill or in pain, and we labeled this behavior “standing head down.” Our data clearly show that the greatest frequency of this behavior was recorded in the FL heifers and cows during every observation period. The lowest percentage of animals standing head down was recorded in the PR heifers and cows, with the other treatments intermediate (for heifers) or generally no different to PR (for cows), and the frequency of expression decreased over time. Thus, standing head down met the description by Mellor et al. (2000) of a useful indicator of pain, as it was seen to a greater extent in treated animals than in controls, and its incidence declined over time as the pain or discomfort receded. It is noteworthy that this indicator suggests that both heifers and cows were experiencing pain or discomfort from FL and DOT spaying to at least d 3 postprocedures. Furthermore, this indicator suggests pain or discomfort in the EIM heifers and cows on d 1 to 3 postprocedures. Electroimmobilizers act by causing muscle contraction/tentany (Molony, 1986; Rushen, 1986) and it has been suggested that tetanic contraction can stimulate nociceptors, particularly in the muscles, inducing pain, and that there may be delayed effects lasting for a few days which may be observed as a reluctance to move (Molony, 1986). It has been suggested that decreases in feed consumption and a lack of interest in food and water can indicate pain (Loeffler, 1986; Cook, 1996), although, of course, there could also be other reasons for a reduction in feeding and drinking. Suppression of feeding has been reported to be indicative of pain in numerous studies of painful conditions and husbandry procedures in cattle (e.g., dehorning of calves, Graf and Senn, 1999; McMeekan et al., 1999; castration of bulls, Fisher et al., 2001; and lameness in dairy cows, Almeida et al., 2008). The lowest percentage of cattle feeding at the end of the d 0 was observed in the FL heifers, followed by the DOT heifers, with no difference among the other treatments. For the cows, the lowest proportions were in the FL, DOT, and EIM treatments. These results again support our conclusions that spaying and electroimmobilization are painful, with the latter being more painful for cows than heifers, perhaps due to their greater muscle mass. The FL heifers, but not cows, showed reduced ruminating at the end of the d 0. This may have been a consequence of the reduced feeding by these cattle, but other studies have reported reduced rumination associated with pain (Sylvester et al., 2004; Almeida et al., 2008; Kolkman et al., 2010). The relatively high proportion of FL heifers drinking at the end of d 0 may have indicated dehydration. Drinking was unlikely to have been related to feeding, as the FL heifers showed the lowest percentage for feeding. It is possible that drinking was a consequence of greater blood loss (internal bleeding) associated with this treatment than the others. The FL cows also showed a relatively high level of drinking at this time, but so, too, did the PR cows. Possibly, the cows were better able to tolerate blood loss than the heifers, as a consequence of their larger body size and greater fluid reserves. Standing and lying are difficult to interpret with regard to pain; some researchers suggest that a reluctance to move may be indicative of pain (Molony et al., 1995; Stafford and Mellor, 2005), whereas others suggest that cattle in pain are more restless (e.g., Mellor et al., 1991). Lying could, however, be indicative of fatigue (Knowles and Warriss, 2007). A greater percentage of FL and DOT heifers were seen lying at the end of d 0 compared with other treatments, but for cows the greatest percentage was in the PR treatment. It is unclear why there were differences between the cows and heifers. The self-licking we recorded in the FL cattle was mostly directed at the wound, and this probably accounted for the greater percentage of FL cattle performing it compared with the other treatments. Self-care of a painful region is reported to be indicative of chronic pain (Zimmermann, 1986), and this fits with the timeframe (3 d post-FL spaying) of increased concentrations of haptoglobin, which suggests pain associated with inflammation of the wounds. Although our results suggest that the FL spayed cattle experienced pain for at least 3 d postprocedure, it was of insufficient duration and/or magnitude to adversely affect BW gains. These findings contrast with other studies that have shown that BW gains of spayed heifers are lower than nonspayed controls in the weeks and months postprocedure (e.g. Jeffery et al., 1997; Jubb et al., 2003; McCosker et al., 2010). In summary, using a combination of recognized, scientific indicators of pain and stress we have shown that, for both heifers and cows, FL and DOT spaying cause similar levels of acute pain. There was, however, evidence from plasma concentrations of bound cortisol, CK, and AST, and serum concentrations of haptoglobin, together with changes in feeding, standing head down, and self-licking behaviors, that FL spaying was associated with longer-term pain and morbidity (prolonged wound healing) that was not found after DOT spaying. Electroimmobilization during FL spaying contributed to the pain and stress of the procedure, as evidenced by increased CK and AST concentrations, and increased standing head down in both heifers and cows, and increased cortisol concentrations and reduced feeding behavior in the cows. Adverse welfare outcomes from spaying were also greater for cows than heifers. Based on our findings we recommend that: i) FL and DOT spaying should not be conducted without measures to manage the associated pain and stress; ii) DOT spaying is preferable to FL spaying; iii) spaying heifers is preferable to spaying cows; and iv) electroimmobilization is painful and stressful and should not be routinely used as a method of restraint. LITERATURE CITED Addis P. B. Nelson D. A. Ma R. T.-I. Burroughs J. R. 1974. Blood enzymes in relation to porcine muscle properties. J. Anim. Sci.  38: 279– 286. Google Scholar CrossRef Search ADS PubMed  Almeida P. E. Weber P. S. D. Burton J. L. Zanella A. J. 2008. Depressed DHEA and increased sickness response behaviors in lame dairy cows with inflammatory foot lesions. Dom. Anim. Endocrinol.  34: 89– 99. Google Scholar CrossRef Search ADS   Blum J. W. Froehli D. Kunz P. 1982. Effects of catecholamines on plasma free fatty acids in fed and fasted cattle. Endocrinol.  110: 452– 456. Google Scholar CrossRef Search ADS   Braun J. P. Lefebvre H. P. Hamliri A. Kessabi M. Toutain P. L. 1993. Creatine kinase in sheep: A review. Rev. Méd. Vétér.  144: 659– 664. Burrow H. M. Seifert G. W. Corbet N. J. 1988. A new technique for measuring temperament in cattle. Proc. Aust. Soc. Anim. Prod.  17: 154– 157. Cook C 1996. Pain in farm animals: Awareness, recognition and treatment. In: Baker R. Einstein R. Mellor D. editors, Farm Animals in Biomedical and Agricultural Research.  ANZCCART, Glen Osmond, Australia. p. 75– 82. de Witte K. Jubb T. Letchford P. 2006. The dropped ovary technique for spaying cattle. In: Australian Cattle Veterinarians Manual. Australian Cattle Veterinarians, Eight Mile Plains,  Australia. p. 14– 20. D'Occhio M. J. Fordyce G. Whyte T. R. Jubb T. F. Fitzpatrick L. A. Cooper N. J. Aspden W. J. Bolam M. J. Trigg T. E. 2002. Use of GnRH agonist implants for long-term suppression of fertility in extensively managed heifers and cows. Anim. Reprod. Sci.  74: 151– 162. Google Scholar CrossRef Search ADS PubMed  Fisher A. D. Knight T. W. Cosgrove G. P. Death A. F. Anderson C. B. Duganzich D. M. Matthews L. R. 2001. Effects of surgical or banding castration on stress responses and behaviour of bulls. Aust. Vet. J.  79: 279– 284. Google Scholar CrossRef Search ADS PubMed  Garcia-Belenguer S. Palacio J. Gascón M. Aceňa C. Revilla R. Mormède P. 1996. Differences in the biological stress responses of two cattle breeds to walking up to mountain pastures in the Pyrenees. Vet. Res.  27: 515– 526. Google Scholar PubMed  GenStat 2008. GenStat for Windows. Release 11.1, 11th ed. VSN Int., Oxford, UK. Graf B. Senn M. 1999. Behavioural and physiological responses of calves to dehorning by heat cauterization with or without local anaesthesia. Appl. Anim. Behav. Sci.  62: 153– 171. Google Scholar CrossRef Search ADS   Habermehl N. L 1993. Heifer ovariectomy using the Willis spay instrument: technique, morbidity and mortality. Can. Vet. J.  34: 664– 667. Google Scholar PubMed  Horadagoda N. U. Knox K. M. G. Gibbs H. A. Reid S. W. J. Horadagoda A. Edwards S. E. R. Eckersall P. D. 1999. Acute phase proteins in cattle: Discrimination between acute and chronic inflammation. Vet. Rec.  144: 437– 441. Google Scholar CrossRef Search ADS PubMed  Jeffery M. Loxton I. Van der Mark S. James T. Shorthose R. Bell K. D'Occhio M. J. 1997. Liveweight gains, and carcass and meat characteristics of entire, surgically spayed or immunologically spayed beef heifers. Aust. J. Expt. Agric.  37: 719– 726. Google Scholar CrossRef Search ADS   Jubb T. Letchford P. 1997. Cattle spaying and electroimmobilisers—Their use in extensive beef cattle herds in the Kimberley region. In: Proc. Australian Cattle Veterinarians Annual Conference,  Brisbane, Australia. p. 31– 35. Jubb T. F. Fordyce G. Bolam M. J. Hadden D. J. Cooper N. J. Whyte T. R. Fitzpatrick L. A. Hill F. D'Occhio M. J. 2003. Trial introduction of the Willis dropped ovary technique for spaying cattle in northern Australia. Aust. Vet. J.  81: 66– 70. Google Scholar CrossRef Search ADS PubMed  Kendall M. Stuart A. Ord J. K. 1983. The Advanced Theory of Statistics, Volume 3, 4th ed. Griffin, London, UK. Knowles T. Warriss P. 2007. Stress physiology of animals during transport. In: Grandin T. editor, Livestock Handling and Transport,  3rd ed. CAB International, Wallingford, UK. p. 312– 328. Kolkman I. Aerts S. Veraecke H. Vicca J. Vandelook J. de Kruif A. Opsomer G. Lips D. 2010. Assessment of differences in some indicators of pain in double muscled Belgian Blue cows following naturally calving vs. caesarean section. Reprod. Dom. Anim.  45: 160– 167. Google Scholar CrossRef Search ADS   Loeffler K 1986. Assessing pain by studying posture, activity and function. In: Duncan I. J. H. Molony V. editors, Assessing Pain in Farm Animals.  Commission of the European Communities, Luxembourg. p. 49– 57. Martin P. Bateson P 1986. Measuring Behaviour. Cambridge University Press, Cambridge, UK. McCosker K. Letchford P. Petherick J. C. Mayer D. McGowan M. 2010. Morbidity, mortality and body weight gain of surgically spayed, yearling Brahman heifers. Aust. Vet. J.  88: 497– 503. Google Scholar CrossRef Search ADS PubMed  McCullagh P. Nelder J. A 1989. Generalized Linear Models, 2nd ed. Chapman and Hall, London. McMeekan C. Stafford K. J. Mellor D. J. Bruce R. A. Ward R. N. Gregory N. 1999. Effects of a local anaesthetic and a non-steroidal anti-inflammatory analgesic on the behavioural responses of calves to dehorning. N. Z. Vet. J.  47: 92– 96. McVeigh J. M. Tarrant P. V. Harrington M. G. 1982. Behavioral stress and skeletal muscle glycogen metabolism in young bulls. J. Anim. Sci.  54: 790– 795. Google Scholar CrossRef Search ADS PubMed  Mellor D. J. Cook C. J. Stafford K. J. 2000. Quantifying some responses to pain as a stressor. In: Moberg G. P. Mench J. A. editors, The Biology of Animal Stress. Basic Principles and Implications for Animal Welfare.  CABI, Wallingford, UK. p. 171– 198. Google Scholar CrossRef Search ADS   Mellor D. J. Molony V. Robertson I. S. 1991. Effect of castration on behaviour and plasma cortisol concentrations in young lambs, kids and calves. Res. Vet. Sci.  51: 149– 154. Google Scholar CrossRef Search ADS PubMed  Molony V 1986. The electroimmobilisation and electroanaesthetisation of farm animals: A review. Report to the Farm Animal Welfare Council, UK. Molony V. Kent J. E. Robertson I. S. 1995. Assessment of acute and chronic pain after different methods of castration of calves. Appl. Anim. Behave. Sci.  46: 33– 48. Google Scholar CrossRef Search ADS   Nakao T. Sato T. Morihoshi M. Kawata K. 1994. Plasma cortisol response in dairy cows to vaginoscopy, genital palpation per rectum and artificial insemination. J. Vet. Med.  41: 16– 21. Google Scholar CrossRef Search ADS   Petherick J. C 2005. Animal welfare issues associated with extensive livestock production: The northern Australian beef cattle industry. Appl. Anim. Behav. Sci.  92: 211– 234. Google Scholar CrossRef Search ADS   Petherick J. C. Doogan V. J. Venus B. K. Holroyd R. G. Olsson P. 2009. Quality of handling and holding yard environment, and beef cattle temperament: 2. Consequences for stress and productivity. Appl. Anim. Behav. Sci.  120: 28– 38. Google Scholar CrossRef Search ADS   Petherick J. C. Holroyd R. G. Doogan V. J. Venus B. K. 2002. Productivity, carcass and meat quality of lot-fed Bos indicus cross steers grouped according to temperament. Aust. J. Expt. Agric.  42: 389– 398. Google Scholar CrossRef Search ADS   Pilz M. Fischer-Tenhagen C. Thiele G. Tinge H. Lotz F. Heuwieser W. 2012. Behavioural reactions before and during vaginal examination in dairy cows. Appl. Anim. Behav. Sci.  138: 18– 27. Google Scholar CrossRef Search ADS   Pinner K. K. R 2006. Lack of animal welfare assessment regarding trans-vaginal spaying of heifers. Can. Vet. J.  47: 266– 272. Google Scholar PubMed  Rowell J. G. Walters R. E. 1976. Analysing data with repeated observations on each experimental unit. J. Agric. Sci.  87: 423– 432. Google Scholar CrossRef Search ADS   Rushen J 1986. Aversion of sheep to electro-immobilization and physical restraint. Appl. Anim. Behav. Sci.  15: 315– 324. Google Scholar CrossRef Search ADS   Stafford K. J. Mellor D. J. 2005. The welfare significance of the castration of cattle: A review. N. Z. Vet. J.  53: 271– 278. Sylvester S. P. Stafford K. J. Mellor D. J. Bruce R. A. Ward R. N. 2004. Behavioural responses of calves to amputation dehorning with and without local anaesthesia. Aust. Vet. J.  82: 697– 700. Google Scholar CrossRef Search ADS PubMed  Zimmermann M 1986. Assessing pain by studying posture, activity and function. In: Duncan I. J. H. Molony V. editors, Assessing Pain in Farm Animals. Commission of the European Communities, Luxembourg. p. 16– 29. Footnotes 1 Heytesbury Beef provided the facilities and cattle for this research; the research was partially funded by Meat & Livestock Australia; Bronwyn Venus (Agri-Science Qld) and Alan McManus (Biosecurity Qld) conducted assays; and these personnel assisted with data collection: Gus Payne (Heytesbury Beef); Trisha Cowley, Annemarie Huey, Harmony James, Gehan Jayawardahana, Andrew Murray, Caroline Smith, and Sarah Streeter (NT Dept Resources); Tracey Longhurst (Agri-Science Qld); and Nancy Phillips (The University of Qld) American Society of Animal Science
TI - Evaluation of the impacts of spaying by either the dropped ovary technique or ovariectomy via flank laparotomy on the welfare of Bos indicus beef heifers and cows
JF - Journal of Animal Science
DO - 10.2527/jas.2012-5164
DA - 2013-01-01
UR - https://www.deepdyve.com/lp/oxford-university-press/evaluation-of-the-impacts-of-spaying-by-either-the-dropped-ovary-CRDwld8M9V
SP - 382
EP - 394
VL - 91
IS - 1
DP - DeepDyve
ER -