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. 2019 Dec 25;98(1):skz386. doi: 10.1093/jas/skz386

Measuring behavioral and physiological responses to pain mitigation for ovariectomy in Bos taurus yearling beef heifers

Jean K Lauder 1,✉, Sonia Marti 2, Karen S Schwartzkopf-Genswein 3, Murray D Jelinski 1, Eugene D Janzen 4
PMCID: PMC6986433  PMID: 31875222

Abstract

Ovariectomy (spaying) using the trans-vaginal dropped ovary technique (DOT) is performed to prevent pregnancy in cull female beef cattle that are not retained for breeding stock in areas practicing extensive grazing management. There are no reports describing analgesia for this surgical procedure. The objective of this study was to measure behavioral and physiological responses to determine whether an analgesic protocol of BXK [butorphanol (0.01 mg/kg), xylazine (0.02 mg/kg), and ketamine (0.04 mg/kg)] injected intramuscularly (i.m.) before spaying could mitigate procedural and immediate postsurgical pain, and whether oral meloxicam (1 mg/kg) administered at the time of spaying could mitigate postsurgical inflammatory pain. Forty-four red Angus and Angus crossbred yearling heifers (322 ± 27.0 kg BW) were randomly allocated to 1 of 3 groups: PALP (control; palpated but not spayed; n = 14), SPAY (spayed with no analgesia; n = 15), and BXKM (spayed with analgesia; n = 15). Behavioral measurements included visual analog scale (VAS) score, flight speed (FS), stride length (SL), and gait score (GS), as well as activity (lying, standing) and feeding behavior. Physiological measurements included salivary cortisol (SC), haptoglobin (Hp), serum amyloid A (SAA), substance P (SP), complete blood count (CBC), and rectal temperature (RT). Saliva and blood samples were collected, and RT, FS, SL, and GS were measured on day −1, day 0 (time of palpation/spaying), and hours 1, 2, 4, and days 1, 2, 4, and 7 after palpation/spaying. The BXKM heifers had lower SC concentrations than SPAY heifers at 1 h (P = 0.01) and 2 h (P = 0.004). Heifers treated with BXKM had Hp concentrations lower than SPAY heifers at 2 d (P = 0.01), 4 d (P < 0.001), and 7 d (P = 0.008), and lower Hp concentrations than PALP heifers at 4 d (P < 0.001). Concentrations of SAA were greater (P = 0.04) in BXKM heifers than in PALP heifers at 1 h and lower in PALP heifers than in BXKM heifers (P = 0.02) and SPAY heifers (P = 0.05) at 1 d. Heifers in the BXKM group had higher RT than PALP and SPAY heifers at 1 h (P < 0.001) and 2 h (P = 0.004). Results suggest that DOT ovariectomy is acutely stressful and painful and administration of BXK before spaying and meloxicam at the time of spaying mitigated the procedural and postsurgical stress, pain, and inflammation.

Keywords: analgesia, animal welfare, anti-inflammatory, beef heifers, ovariectomy, spay

Introduction

Ovariectomy (spaying) prevents pregnancy and improves marketing of cull heifers destined for sale rather than breeding (Rupp and Hamilton, 1995). The trans-vaginal dropped ovary technique (DOT) is considered the least noxious spaying method, but still causes stress and pain (Petherick et al., 2013). The Canadian beef Code of Practice recommends that producers seek advice for analgesia for spaying (National Farm Animal Care Council, 2013); however, no pain mitigation strategy for spaying has been published to date.

Physiological pain indicators in animals can be confounded by stress, but may be more useful than behavioral pain indicators in species such as cattle that are observed by potential predators (Weary et al., 2006). Ovariectomy pain potentially arises from visceral tissue trauma (procedural pain) and the inflammation it provokes (postsurgical pain), both should be addressed for effective pain control. Pre-emptive multimodal therapy using a combination of analgesics, such as BXK (0.01 mg/kg butorphanol, 0.02 mg/kg xylazine, and 0.04 mg/kg ketamine) and meloxicam, delivers synergistically superior analgesia compared with using a single analgesic agent (Gaynor and Muir, 2015). The duration of effect for intramuscular (i.m.) BXK has been reported as <2 h, with a clinical effect onset within 5 min (Abrahamsen, 2013), allowing convenient administration to heifers waiting in the race prior to entering the squeeze chute. The oral nonsteroidal anti-inflammatory drug, meloxicam, provides the longest anti-inflammatory dose duration currently available for cattle (Stock and Coetzee, 2015).

The objective of this study was to determine whether BXK administered pre-emptively (5 min before spaying), and oral meloxicam administered at the time of spaying, mitigated procedural and postsurgical pain. We hypothesized that the periprocedural administration of BXK and meloxicam should provide preventative analgesia for DOT ovariectomy.

Materials and Methods

The Lethbridge Research Centre (LRC) Animal Care Committee (ACC Protocol # 1643) and the University of Saskatchewan Animal Care Committee Animal Research Ethics Board approved this study protocol (UACC AUP # 20160108). The Canadian Council of Animal Care guidelines (Canadian Council on Animal Care, 2009) were followed for the care of the study heifers.

Animals, Animal Management, and Handling Facility

Forty-five commercial red Angus and Angus crossbred yearling heifers (322 ± 27.0 kg) purchased from 2 sources were kept at the Agriculture and Agri-Food Canada Lethbridge Research and Development Centre (LRC, Lethbridge, AB, Canada) for 4 wk before the start of the study to acclimate them to the facility and handling procedures. Heifers were housed in groups of 9 in 5 feedlot pens (21 m × 27 m). Each pen had an ad libitum water supply and a 2.4 m × 24 m concrete apron in front of a feed bunk with a GrowSafe System (GrowSafe Systems, Calgary, AB, Canada) installed to measure individual heifer feed intake and feeding behavior. Heifers were fed a diet of 89.2% barley silage, 8.2% dry rolled barley, and 2.6% standard feedlot supplement to meet nutritional requirements (National Academies of Sciences Engineering and Medicine, 2016). Heifers were moved from their home pens to a handling facility with a hydraulic squeeze chute (Cattlelac Cattle Handling Systems, Reg Cox Feedmixers Ltd., Lethbridge, AB, Canada) for sampling.

On day −1, heifers were fitted with a unique ear tag and a back tag (Regular Back Tags, Wheel‘n’Tree Enterprises, Sherwood Park, AB, Canada) that was affixed using an adhesive (Livestock cement, Wheel‘n’Tree Enterprises) for ease of individual identification during behavioral assessments. Feed was withheld after sampling on day −1 for 20 h to fast heifers before ovariectomy to reduce rumen fill and decrease risk of iatrogenic surgical visceral injury. On day 0, all heifers were administered a prophylactic treatment of 20 mL of 200 mg/mL oxytetracycline (Oxyvet 200 LA, Vetoquinol NA Inc., Lavaltrie, QC, Canada) i.m. in 2 sites (10 mL per site). Heifers were monitored daily for morbidity and mortality.

Experimental Design and Treatments

Heifers were blocked by body weight and randomly assigned to 1 of 3 treatment groups so there were 3 heifers from each treatment group in each pen: 1) PALP (positive control/sham ovariectomy group; n = 14): reproductive tract palpated rectally for 1 min and ovariotome introduced vaginally without creating a colpotomy incision, sterile diluent injection (Sterile Water USP, Bimeda Canada, Cambridge, ON, Canada) administered i.m. 5 min before palpation and oral water drench administered immediately before palpation; 2) SPAY (negative control group; n = 15): ovariectomized without analgesia, sterile diluent injection administered i.m. 5 min before ovariectomy and oral water drench administered immediately before ovariectomy; 3) BXKM (treatment group; n = 15): ovariectomized and administered a combination of butorphanol [0.01 mg/kg (Torbugesic, Zoetis Canada Inc., Kirkland, QC, Canada)], xylazine [0.02 mg/kg (Rompun 20 mg/mL Injectable, Bayer Inc., Mississauga, ON, Canada)], and ketamine [0.04 mg/kg (Narketan, Vetoquinol N.A. Inc.)] injection administered i.m. 5 min before ovariectomy, and oral meloxicam [1 mg/kg (Meloxicam Oral Suspension, USP, Solvet, Alberta Veterinary Laboratories Ltd., Calgary, AB, Canada)] administered immediately before ovariectomy. The treatment administrator was blinded to the treatment group.

On day 0, either BXK or sterile diluent was injected i.m. in the neck of each heifer using an 18-gauge 2.5-cm stainless steel needle (Ideal D3 Detectable Needles, Animal Safety Division, Neogen, Lexington, KY) while restrained in the chute before entering the squeeze. A digital timer was used to verify that a minimum of 5 min had elapsed between injection and palpation or ovariectomy. An oral drench of either meloxicam or water was administered before palpation or spaying. A veterinarian experienced in palpating dairy cattle ovariectomized the heifers with a Willis ovariotome using the DOT as described by Habermehl (1993). The veterinarian was instructed to rectally palpate heifers in the PALP group for 1 min with similar effort as the heifers being ovariectomized.

Sample Collection Schedule

Each heifer had blood and saliva samples taken, and rectal temperature recorded (GLA M750 Livestock Thermometer, San Luis Obispo, CA) on the day before spaying/palpation (day −1), at the time of spaying/palpation (day 0), at 1, 2, and 4 h after spaying/palpation, and on days 1, 2, 4, and 7 post-spaying/palpation. Visual analog scale (VAS) scores and pain-related behaviors were recorded at the time of spaying (day 0). Body weight was recorded with a digital scale on day −1, day 0, day 7, and day 29. Feeding and standing/lying behavior was recorded continuously from day −1 to day 7. Videos of heifers in their pens were recorded starting 4 h after ovariectomy for 5 h on day 0, and 24 h later for 4 h on day 1. Hair samples were gathered on day −1 and day 29, and final body weight was recorded and 10 mL of jugular blood was collected in a nonadditive tube for estradiol concentration on day 29.

Procedures for Behavioral Measurements of Pain

VAS score

While in the squeeze chute for ovariectomy/palpation, 2 trained observers blinded to treatment group marked their subjective assessment of the degree of pain experienced by the heifers during the procedure on a 10-cm horizontal line as previously described by Moya et al. (2014). The distance from the left end of the line to the observer’s mark, to the nearest 0.5 cm, was the VAS score, with a larger score indicating more severe pain.

Pain-related behaviors during ovariectomy

Pain-related behaviors (falling attempts, kicking/leg lifts, tail flicks, body flinches, head movements, and vocalization) were recorded during ovariectomy/palpation by both observers.

Flight speed

Flight speed (FS) was the time taken for a heifer released from the squeeze chute to travel between 2 pairs of light beam generators and reflectors (“electric eyes”; Polaris Multi-Event Timer, FarmTek, Inc., Wylie, TX), previously described by Baldridge et al. (2011), with a modification in distance between electric eyes. The first electric eye was placed approximately 1 m in front of the squeeze chute head gate, at approximately 0.9 m in height, and 2.5 m from the second pair. Transit time between beams was converted from seconds to speed (m/s).

Stride length and gait score

After exiting the squeeze chute, heifers traveled in a straight line on their way to their home pen past a video camera (Panasonic WVCP474; Panasonic Canada, Inc., Mississauga, ON, Canada) set beside the alleyway for measuring stride length (SL) on day −1, day 0 (immediately and 1, 2, and 4 h after spaying/palpating) and on days 1, 2, 4, and 7 after spaying/palpating, as previously described by Currah et al. (2009), with some modifications. A 1.003-m-long fence panel was used as the measuring reference. Two sequential pictures of each heifer while her hind feet were in contact with the ground and alternate hind limbs placed forward were captured with GOM player software (GOMlab, Gretech Corporation, Seoul, South Korea). The stride distance from the midpoint of each hind foot was measured using ImageJ software (Research Services Branch, National Institute of Mental Health, Bethesda, MD), and the average of the 2 stride distances was recorded as the SL measurement. Heifers that ran past the camera, or those whose feet could not be seen clearly, could not be measured, and were treated as missing data points. Video images used for SL were used to categorize the gait used by heifers leaving the squeeze chute. Gait score (GS) was scored as walk, trot, or run.

Feeding behavior and ADG

Each pen feed bunk was outfitted with an electronic feed bunk monitoring system (GrowSafe System, Airdrie, AB, Canada) used to continuously record individual heifer feeding behavior (Schwartzkopf-Genswein et al., 1999). The system detected individual radio frequency identification ear tags when heifers were within 50 cm of the load cell. Logged data were used to determine mean daily feed intake (kg/d), feeding time (min/d), feeding rate (g/min), meal frequency (number/d), meal duration (min/meal), and meal size (kg/meal). A meal criterion of 85 s was used as previously described by Schwartzkopf-Genswein et al. (1999). Body weight was recorded for individual heifers while in the squeeze chute on day −1, day 0, day 7, and day 29 for sampling to determine ADG from day 0 to day 7 and from day 0 to day 29.

Pain-related behaviors after ovariectomy

On day −1, accelerometers for monitoring standing and lying behavior (HOBO Pendant G Data Logger, Onset Computer Corporation, Bourne, MA) were protected with waterproof padding and covered with self-adhesive bandaging material (Flexible Wrap, Professional Preference, McCarthy and Sons Service, Calgary, AB, Canada) and then secured vertically to the lateral right hind metatarsus of each animal using self-adhesive bandaging material. Vertical (X) and horizontal (Z) axis orientation data were recorded for 7 d in 24-h periods and reported standing/lying time budgets (daily percentage), mean standing and lying duration (min/d), and standing and lying bouts (number/d) as validated by the University of British Columbia Animal Welfare Program (UBC AWP, 2013). Accelerometers were removed on day 7, and the contained data were downloaded to a computer for analysis.

Heifers were video recorded for 5 h on day 0 after the 4 h post-ovariectomy samples were collected and for 4 h on day 1 to record behaviors (walking, standing, lying in sternal recumbency, lying in lateral recumbency, tail flicking, grooming, foot stamping, back arching, and tail lifting).

Procedures for Physiological Measurements of Stress and Pain

CBC

Jugular blood samples were taken from each heifer with an 18-gauge needle into two 6-mL EDTA tubes and one 10-mL nonadditive tube (BD, Franklin Lakes, NJ). One 6-mL EDTA tube was used for CBC analysis using a HemaTrue Hematology Analyzer (Heska, Loveland, CO) to yield total white blood cell (WBC) count, lymphocyte, monocyte and granulocyte concentrations, hematocrit (HCT), total red blood cell (RBC) count, and total platelet (PLT) count. Neutrophil:lymphocyte (N:L) ratio estimates were calculated using the granulocyte and lymphocyte concentration data.

Salivary cortisol

Saliva samples were obtained from the oral cavity using cotton swabs, which were placed in plastic tubes and stored on ice, then frozen within 2 h at −20 °C until assayed for cortisol concentration using a commercial competitive immunoassay kit according to the manufacturer’s instructions (Salimetrics Cortisol Enzyme Immunoassay Kit, Salimetrics, LLC., State College, PA). Samples were assayed in duplicate, and cortisol concentration was determined from a linear standard curve generated from kit standards ranging from 0.12 to 30 ng/mL. Interassay and intra-assay CVs were 29.64% and 7.36%, respectively.

Hair cortisol

Hair from the forehead of each heifer was shaved on day −1 and day 29 using electric clippers, placed in a labeled plastic bag and stored for cortisol analysis. From each sample, 250 mg of hair was washed twice with isopropanol, dried, and minced in a 10-mL metal cylinder containing a 12-mm metal ball and ground with a mixer mill (MM 200, Retsch Inc., Newtown, PA) for 1 min and 40 s at a vibrational frequency of 1,320 per min. A measure of 50 mg of minced hair in a 5-mL glass vial was used for cortisol extraction and assayed by modifying the method described by Moya et al. (2013). Briefly, after adding 1.5 mL of methanol, and incubating for 18 h on a shaker (GYROMAX Benchtop Incubator Shaker, Amerex Instruments, Inc., Concord, CA) at 30 °C and 100 rpm, 0.75 mL of supernatant was pipetted into a 1.5-mL microtube and evaporated at 45 °C under a 15 kPa nitrogen stream in a fume hood. One hundred microliters of phosphate-buffered saline was used to reconstitute each sample and the same commercial kit used for salivary cortisol was used for hair cortisol determination. Interassay CV was 15.92%, and intra-assay CV was 7.82%.

Substance P

The second set of 6 mL EDTA tubes had benzimidine hydrochloride (1 mM/mL of blood) added to minimize substance P (SP) degradation, then were centrifuged at 1.5 × g for 15 min at 0 °C and the plasma decanted and frozen at −80 °C. These samples were sent to Iowa State University (Ames, IA) for SP analysis using immunoassay as described by Coetzee et al. (2008). Interassay and intra-assay CVs were 17.07% and 8.84%, respectively.

Acute phase proteins: haptoglobin and serum amyloid A

Serum was harvested from the 10-mL nonadditive tubes by centrifuging at 1,600 × g and 4 °C for 15 min, transferred to labeled tubes and frozen at −20 °C in triplicate. One-half milliliter samples were sent to the University of Guelph (Guelph, ON, Canada) for haptoglobin (Hp) analysis with a Roche Cobas c501 biochemistry analyzer (Roche Diagnostics, Laval, QC, Canada) using Tridelta bovine Hp calibrator reagents (TP-801-CAL, Tridelta Development Ltd., Maynooth, County Kildare, Ireland) and 2vente levels of pooled bovine serum controls. Interassay CV for Hp analysis was 3.57%. Duplicate serum samples were assayed for serum amyloid A (SAA) using a commercial solid phase sandwich ELISA kit (Tridelta Phase Range Multispecies SAA ELISA kit, Tridelta Development Ltd.). The interassay CV for SAA was 15.50%, and intra-assay CV was 8.46%.

Hormonal assay: estradiol

Triplicate serum samples from each heifer were decanted from 10-ml nonadditive tubes of blood samples obtained on day 29 and were sent to the University of Saskatchewan (Saskatoon, SK, Canada) for estradiol (E2) concentration determination using an extracted radioimmunoassay technique performed using prepared standards in charcoal stripped serum (Joseph et al., 1992). Interassay and intra-assay CVs for E2 were 21.3% and 11.8%, respectively.

Statistical analysis

The individual heifer was the experimental unit. One heifer with epistaxis was removed from the trial on day −1, leaving 14 heifers in the PALP group and 15 heifers each in the SPAY and BXKM groups. Normal data distribution was tested before analysis using the PROC UNIVARIATE procedure (SAS, Version 9.4, SAS Institute Inc., Cary, NC), and outliers were identified with box plots. Normally distributed data were analyzed using the PROC MIXED repeated measures procedure in SAS where day −1 values were used as covariates, except for the bodyweight covariate for calculating ADG, where day −1 and day 0 bodyweights were averaged because heifers were fasted after day −1 sampling. Flight speed from day −1 was also used as a covariate to compare results obtained to determine if heifer temperament may have been a confounder for SL, accelerometer data, feeding behavior, and physiological indicators. Time and treatment were considered fixed effects and tested for interactions, whereas heifer within pen was a considered a random effect. Time was considered a repeated factor, and for each variable, 3 variance−covariance structures were applied (compound symmetry, autoregressive order 1, and unstructured) to select a covariance structure for the heifer within pen (the error term). The analysis used was the most appropriate covariance structure that minimized Schwarz’s Bayesian information criterion. When tendencies or significant interactions were present, post hoc PDIFF testing was used to generate P-values for least squares means (LSM) differences. Physiological data were natural log transformed, and pen behavioral data were square root transformed to achieve normal distribution before analysis. Nontransformed LSM with transformed SEM were reported. When transformation was unable to normalize data, VAS scores were analyzed with the nonparametric Wilcoxon matched-pairs signed-ranks test using Stata statistical software (version 14.2, StataCorp, College Station, TX) to assess interobserver agreement. Intraclass correlation coefficient was estimated using a 2-way, mixed effect mode in Stata to determine consistency of agreement between observers for individual heifer VAS scores. The Kruskal−Wallis rank sum test for nonparametric data using Stata was used to determine whether mean VAS score ranks were the same between treatment groups, and to compare mean estradiol concentration ranks between groups on day 29. Two-way tables with Fisher’s exact test in Stata were used for GS and treatment group comparisons of pain-related behaviors during ovariectomy. Results were considered significant when P ≤ 0.05, and tendencies were declared when 0.05 < P ≤ 0.10.

Results and Discussion

Morbidity and Mortality

Study heifers had a 0% morbidity and mortality rate. McCosker et al. (2010) cited morbidity rates for DOT ovariectomy of 0.7% with affected heifers being “depressed, slow to rise or walking with restricted gait” for 1 to 4 d. In that study, 9% of heifers demonstrated elevated tail carriage for up to 2 h post-spaying, with 1.5% showing this behavior intermittently for 2 d. No differences in tail carriage were observed in our study, but observations could not be made while heifers were moved through the handling system for physiological sampling. McCosker et al. (2010) reported a mortality rate of 0.5% occurring within 4 d of spaying (including 1 pregnant heifer), and 1.5% occurring 10 to 21 d after spaying without using antibiotics; the latter mortalities were likely infectious in origin, as another animal showed clinical signs of tetanus. All heifers in the present study were vaccinated against Clostridial disease and administered oxytetracycline prophylactically.

Behavioral Measurement Results

VAS score

Consistency of agreement between observers for individual heifer scores was moderately high (71%). There were no differences for each of the observers in VAS scores between treatments (P = 0.33, observer 1; P = 0.56, observer 2); therefore, VAS scores were combined, yielding mean VAS scores of 0.52 ± 0.633, 0.74 ± 0.923, and 0.67 ± 0.551 cm for PALP, SPAY, and BXKM groups, respectively, supporting the previously reported notion that behavior exhibited during spaying appears to be similar to rectal palpation (Habermehl, 1993). Spaying appears less noxious than castration, as mean VAS scores of 2.7 to 3.3 cm were reported in castrated Angus bull calves treated with meloxicam (Meléndez et al., 2017a). Our study heifers were restrained in a hydraulic chute with their tails held vertically during palpation/spaying, which may have reduced their ability to freely display pain-related behaviors. Small group size may have been a factor in the inability to detect behavioral differences; however, in an acute pain castration study, groups of 10 to 12 calves were sufficiently large to detect differences in VAS scores (Meléndez et al., 2017b).

Pain-related behaviors during ovariectomy

There were no differences between groups for falling attempts, kicking/leg lifting, tail flicks, body flinches, head movement, or vocalization (P ≥ 0.12; data not shown). Behavior of DOT spayed heifers has been described as “largely unaffected” by the procedure in the days after ovariectomy (Habermehl, 1993; Jubb et al., 2003). It has been proposed that as prey animals, cattle are more inclined to conceal behavioral indicators of pain to avoid attracting predator attention (Weary et al., 2006; Coetzee, 2011). The lack of difference between treatments for pain-related behaviors recorded during ovariectomy may reflect the nature of the tissue trauma caused by DOT spaying, which is limited only to visceral tissue, unlike castration that traumatizes both superficial and visceral tissues. Visceral pain is conveyed indirectly via the spinoreticular tract to the cerebral cortex where pain is perceived, unlike superficial pain that reaches the cerebral cortex directly via the spinocervicothalamic tract (Hellyer et al., 2007). Large overlapping nociceptor fields, a large component of “silent” nociceptors recruited only during inflammation (Gaynor and Muir, 2015), and a larger proportion of dull pain-conveying C fibers compared with sharp pain-conveying Aδ fibers (Hellyer et al., 2007) provide explanations for few visceral pain-related behaviors during DOT ovariectomy. Estrogen also affects pain by increasing size and sensitivity of pelvic nociceptors and activating nearby pain sensitizing N-methyl-D-aspartate (NMDA) receptors (Sengupta, 2009), so individual heifers in an unsynchronized group probably experience some pain variability when spayed.

Flight speed

A treatment × time interaction (P = 0.03) was identified for FS, with mean ± SEM FS recorded as 2.4 ± 0.25, 2.2 ± 0.24, and 2.7 ± 0.24 m/s, respectively, for PALP, SPAY, and BXKM heifers. At 24 h after ovariectomy/palpation, heifers in the BXKM group exhibited faster (3.0 ± 0.24 m/s; P = 0.02) FS compared with PALP heifers (2.1 ± 0.25 m/s), and tended to be faster (P = 0.08) than SPAY heifers (2.0 ± 0.24 m/s), which suggests that the BXKM heifers may have felt more comfortable when the oral meloxicam was near its 12 h postadministration therapeutic peak (Coetzee et al., 2009). Baldridge et al. (2011) reported that Holstein calves given i.m. BXK immediately prior to castration had reduced FS compared with placebo-treated controls; however, the BXK dosage used was approximately double the dosage used in our study and resulted in more profound sedation that was sufficient to cause a calf exiting the chute to become recumbent.

Stride length and gait score

There were no treatment or treatment × time interaction effects for heifer SL and no difference in LSMs between groups (Table 1). There were no differences in GS between treatment groups at any of the sampling times (P ≥ 0.18; data not shown). Calf SL has been used to measure pain, as castration can cause shortened SL (Currah et al., 2009). The absence of differences in SL and GS between treatments in our study suggests that they may not be suitable indicators of visceral pain associated with DOT ovariectomy or that the pain was not severe enough to affect these behaviors.

Table 1.

Pain-related behaviors of yearling heifers after palpation (PALP; n = 14), ovariectomy1 without analgesia (SPAY; n = 15) and ovariectomy1 with analgesia (BXKM2; n = 15)

Treatment P-value
Item PALP SPAY BXKM SEM T Time T × Time
Stride length3, cm Accelerometer data4 59.5 59.7 63.2 1.50 0.14 <0.001 0.72
 Standing, % 46.5 48.7 49.3 0.98 0.16 <0.001 0.96
 Standing duration, min 52.5b 55.7ab 59.7a 2.42 0.10 <0.001 0.63
 Lying duration, min 49.3 50.8 48.3 2.83 0.81 <0.001 0.86
 Standing bouts, n 13.3 13.0 12.5 0.49 0.44 <0.001 0.69
 Lying bouts, n 16.2 15.2 15.7 0.85 0.68 <0.001 0.78
Behavior 4 h after treatment5
 Walking, min 11.4 10.5 13.1 0.60 0.45 — —
 Standing, min 98.4 73.7 89.9 1.85 0.43 — —
 Lying, min 187.4 191.1 158.7 1.83 0.65 — —
  Lying (sternal), min 187.4 187.7 156.4 1.78 0.67 — —
  Lying (lateral), min 0.0 3.4 2.3 0.53 0.46 — —
 Tail flicking, n 162 70 117 2.3 0.31 — —
 Grooming, n 6.8 11.0 6.6 0.99 0.95 — —
 Foot stamping, n 4.6 5.6 5.0 0.51 0.90 — —
 Back arching, n 0.4 0.8 0.2 0.13 0.44 — —
 Tail lifting, n 4.6 6.4 4.4 0.44 0.63 — —
Behavior 24 h after treatment6
 Walking, min 10.4 11.0 15.2 0.28 0.17 — —
 Standing, min 20.9 22.0 30.5 0.40 0.17 — —
 Lying, min 128.8c 84.5d 60.3d 0.86 0.01 — —
  Lying (sternal), min 128.8e 84.3f 60.3f 0.86 0.01 — —
  Lying (lateral), min 0.0 0.2 0.0 0.38 0.20 — —
 Tail flicking, n 183 324 229 2.9 0.58 — —
 Grooming, n 2.9 4.7 9.7 0.70 0.55 — —
 Foot stamping, n 3.6 5.8 9.0 0.42 0.25 — —
 Back arching, n 0.4 0.3 0.7 0.13 0.68 — —
 Tail lifting, n 7.4 5.5 4.2 0.48 0.23 — —
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1Ovariectomies were performed using the dropped ovary technique (DOT).

2BXKM analgesic protocol used consisted of an injectable combination of butorphanol (0.01 mg/kg), xylazine (0.02 mg/kg), and ketamine (0.04 mg/kg) (BXK) administered i.m. 5 min before ovariectomy, and meloxicam (1 mg/kg) administered orally at the time of ovariectomy.

3Stride length was measured at the time of palpation/ovariectomy (da 0) and at 1, 2, 4, 24, 48, 96, and 168 h after palpation/ovariectomy.

4Accelerometer data were analyzed from day of palpation/ovariectomy (day 0) to day 7 after palpation/ovariectomy to generate least squares means (LSM; ± SEM) for standing and lying behavior measurements.

5Pain-related behaviors were tallied using recorded video starting at 4 h on day 0 (time of palpation/ovariectomy; 5 h of video observed).

6Pain-related behaviors were tallied using recorded video 24 h after ovariectomy (4-h video observed).

a−fLSMs within the rows with dissimilar superscripts differed (P ≤ 0.05).

Feeding behavior and ADG

No treatment differences in feeding behavior (Table 2) or ADG (Table 3) were observed between day 0 to day 7. However, over the 29 d interval, SPAY heifers tended to have lower ADG (P = 0.10) than PALP heifers (Table 3), which is consistent with the findings of Petherick et al. (2011) and Jubb et al. (2003) who reported lower BW in DOT spayed heifers compared to unspayed controls at 42 and 60 d, respectively. Pain in animals is recognized as a reason for reduced feed intake (Weary et al., 2006). Petherick et al. (2013) observed fewer spayed heifers feeding than controls on the day of DOT ovariectomy, but not in the 3 d after spaying; however, feed intake monitoring revealed no differences in feeding behavior between treatments in our study. Pen confinement may have reduced our study heifers’ ability to exhibit differences in activity and feeding, as feed and water were both close at hand.

Table 2.

Feeding behavior (least squares means ± SEM) of yearling beef heifers palpated (PALP; n = 14), ovariectomized1 without analgesia (SPAY; n = 15) and ovariectomized1 with analgesia (BXKM2; n = 15) from day 0 to day 7 post-procedure

Treatment P-value
Item PALP SPAY BXKM SEM T3 Time T × Time
Feed intake, kg 12.3 12.2 11.5 0.47 0.12 <0.001 0.92
Feeding time, min 220.8 225.5 212.1 8.69 0.28 <0.001 0.98
Feeding rate, g/min 56.8 56.3 54.4 1.27 0.32 <0.001 0.64
Meal4 frequency, n 14.3 14.8 14.1 0.47 0.60 <0.001 0.86
Meal duration, min 16.4 17.0 16.5 0.87 0.80 <0.001 0.97
Meal size, kg/meal 0.92 0.93 0.86 0.005 0.27 <0.001 0.77
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1Ovariectomies were performed using the dropped ovary technique (DOT).

2BXKM analgesic protocol used consisted of an injectable combination of butorphanol (0.01 mg/kg), xylazine (0.02 mg/kg), and ketamine (0.04 mg/kg) (BXK) administered i.m. 5 min before ovariectomy, and meloxicam (1 mg/kg) administered orally at the time of ovariectomy.

3T: treatment (PALP, SPAY, or BXKM).

4Meal was defined at 85 s or more of time heifer was detected at the feed bunk.

Table 3.

Least squares means (± SEM) for BW and ADG of yearling beef heifers after palpation (PALP; n = 14), ovariectomy1 without analgesia (SPAY; n = 15) and ovariectomy1 with analgesia (BXKM2; n = 15) from day 0 to day 7 and day 0 to day 29 post-procedure

Treatment P-value
Item PALP SPAY BXKM SEM T
Initial BW3, kg 315 315 314 4.3 0.99
BW on day 7, kg 328 326 325 3.2 0.60
ADG (day 7), kg/d 1.94 1.58 1.51 0.440 0.62
Final BW (day 29), kg 374a 362b 365 4.7 0.03
ADG (day 29), kg/d 1.77c 1.34d 1.45d 0.16 0.03
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1Ovariectomies were performed using the dropped ovary technique (DOT).

2BXKM analgesic protocol used consisted of an injectable combination of butorphanol (0.01 mg/kg), xylazine (0.02 mg/kg) and ketamine (0.04 mg/kg) (BXK) administered i.m. 5 min before ovariectomy, and meloxicam (1 mg/kg) administered orally at the time of ovariectomy.

3Initial BW was the average of day −1 (pre-fasting BW) and day 0 (post-fasting BW).

a−dLSMs within the rows with dissimilar superscripts differed (P ≤ 0.05).

Pain-related behaviors after ovariectomy

There was a treatment tendency effect for standing duration (P = 0.10; Table 1), as the BXKM heifers stood for longer durations (P = 0.03) than PALP heifers, which may support the efficacy of the analgesic protocol; however, pen confinement may have limited the opportunity for the heifers to display differences in activity. There were no differences between groups for pain-related behaviors (tail flicking, grooming, foot stamping, back arching, and tail lifting) on the video recordings obtained on day 0. However, on day 1 after palpation/ovariectomy a treatment effect (P = 0.01) was observed as BXKM and SPAY heifers spent less time lying in sternal recumbency than the PALP heifers (P = 0.003 and P = 0.04, respectively; Table 1). Pain is recognized as a reason for reduced activity in animals (Weary et al., 2006), but the decreased time that the study heifers spent in sternal recumbency may be a reflection of visceral discomfort after ovariectomy. These results contrast with those of Petherick et al. (2013), who reported that more DOT spayed heifers lay in sternal recumbency on the day of ovariectomy, but not in the ensuing 3 d. Our study heifers received no anabolic implants to avoid confounding activity measurement by the mounting behavior associated with their administration, although implanting is a common procedure to improve performance after ovariectomy in North America (Rupp and Hamilton, 1995). Overall, behavioral differences in activity including feed intake in DOT ovariectomized heifers may be unsuitable for pain measurement purposes or the pain from ovariectomy is either not severe enough or of long enough duration to cause a measurable change in these behaviors.

Physiological Measurement Results

CBC

A treatment × time effect (P < 0.004) was observed in the RBC counts between treatment groups (Table 4). The BXKM heifers had lower RBC counts than PALP heifers post-spaying at 24 h (P < 0.001) and 48 h (P = 0.04) and SPAY heifers tended to have lower RBC counts than PALP heifers at 24 h post-spaying (P = 0.09; Fig. 1), that likely reflected the internal blood loss associated with ovariectomy; however, the differences were clinically insignificant. Platelet counts were not different between treatment groups (P = 0.79), suggesting that meloxicam did not adversely affect hemostasis. Meloxicam is considered to be more specific to blocking the COX-2 enzyme and therefore is less antithrombogenic than NSAIDs that are more COX-1 specific (Babos et al., 2013). However, splenic contraction from stress induced epinephrine release may also play a role in maintaining the platelet count within normal limits (Roland et al., 2014). There was a treatment × time effect (P < 0.001) for WBC count (Table 4); at 2 h postspaying SPAY heifers had greater WBC counts than BXKM heifers (P = 0.02) and 4 h post-spaying both SPAY and BXKM heifers had greater WBC counts than PALP heifers (P < 0.001; Fig. 1), which may reflect a stress neutrophilia associated with increased cortisol release in the SPAY heifers and a smaller delayed cortisol release in the BXKM heifers. No treatment × time effect differences were observed for N:L ratios between groups (P = 0.19; Table 4). Stress can cause an increased N:L ratio, but the N:L ratio for all groups was greater than the normal ratio of 0.5 reported by Roland et al. (2014) for health adult dairy cows, and may reflect the role of handling stress in beef heifers. Infection, trauma-induced chronic inflammation and stress are the most common causes of increased WBC counts in cattle (Roland et al., 2014). The brief leukocytosis in the SPAY heifers suggests that DOT ovariectomy did not induce chronic inflammation or stress and the even shorter period of leukocytosis in the BXKM heifers suggests that the pharmaceuticals may have reduced the inflammation and stress of ovariectomy.

Table 4.

Least squares means (LSM) (± SEM) of physiological indicators of stress and pain in yearling beef heifers after palpation (PALP; n = 14), ovariectomy without analgesia (SPAY; n = 15) and ovariectomy with analgesia (BXKM; n = 15)

Treatment P-value
Item1 PALP SPAY BXKM SEM T Time T × Time
Rectal temperature, °C 39.7 39.7 39.8 0.08 0.32 <0.001 <0.001
Salivary cortisol, nmol/L 7.8b 10.5a 7.0b 0.12 0.08 <0.001 0.07
Substance P, pg/mL 78.7 79.8 78.6 0.04 0.87 <0.001 0.93
Haptoglobin, g/L 0.46b 0.73a 0.43b 0.11 0.02 <0.001 <0.001
Serum Amyloid A, µg/mL 118b 141a 142a 0.10 0.21 <0.001 0.02
Hair cortisol2, pg/mL 3.1 3.2 3.5 0.12 0.90 — —
Complete blood cell count
 Red blood cells, 106/µL 8.1a 7.9 7.9b 0.10 0.28 <0.001 <0.01
 Platelet count, 103/µL 393 385 378 9.20 0.52 <0.001 0.79
 White blood cells, 103/µL 10.4b 10.8a 10.6a 0.29 0.50 <0.001 <0.001
 N:L ratio 0.77 0.87 0.76 0.07 0.35 <0.001 0.19
Estradiol3, pg/mL 1.4 1.0 1.1 0.23 0.40 — —
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1Heifers had saliva and blood samples collected at the time of palpation/ovariectomy (day 0) and at 1, 2, 4, 24, 48, 96 and 168 h after palpation/ovariectomy.

2Hair samples were taken on day 0 and day 29.

3Blood sample for estradiol was taken on day 29.

a−dLSMs with differing superscripts within a row differ (P ≤ 0.05).

Figure 1.

Figure 1.

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Least squares means (± SEM) for RBC (top) and WBC (bottom) counts in yearling beef heifers after palpation (PALP; blue line), ovariectomy without analgesia (SPAY; red line) and ovariectomy with analgesia (BXKM; green line) on day 0, at 0 (time of palpation/ovariectomy), 1, 2, 4, 24, 48, 96 and 168 h after palpation/ovariectomy. BXKM analgesia consisted of BXK (butorphanol 0.01 mg/kg, xylazine 0.02 mg/kg, and ketamine 0.04 mg/kg) injected i.m. 5 min before ovariectomy and meloxicam (1 mg/kg) administered orally at the time of ovariectomy. Differing letters over sampling time points indicate differences between groups (P ± 0.05).

Salivary and hair cortisol

A treatment effect tendency (P = 0.08; Table 4) and a treatment × time interaction tendency (P = 0.09) were observed for salivary cortisol concentrations, as SPAY and PALP heifers had greater concentrations than BXKM heifers (P = 0.01 and P = 0.03, respectively; Fig. 2) at 1 h after spaying/palpation. At 2 h after ovariectomy, SPAY heifers had greater salivary cortisol concentrations than BXKM heifers (P = 0.005) and at 4 h after ovariectomy, both SPAY and BXKM heifers had greater concentrations (P = 0.02 and P = 0.03, respectively) than PALP heifers. There were no differences between treatment groups on days 1, 2, 4, and 7 after spaying/palpation (P > 0.10). Cortisol is the primary hormone responsible for the stress response. Although not a direct measure of pain, cortisol concentration has been recognized as corresponding to the degree of noxiousness of a procedure; however, it is also affected by handling stress (Mellor and Stafford, 2000; Coetzee et al., 2010; Coetzee, 2011). Stress simulation by ACTH administration in Holstein calves caused peak salivary cortisol concentrations at 40 min post-administration, with a gradual return to baseline by 360 min (Negrão et al., 2004). Unlike Petherick et al. (2013), which reported no difference in free plasma cortisol concentrations in Bos indicus heifers that were DOT spayed without analgesia compared to unspayed controls, SPAY heifers developed a typical salivary cortisol response to surgery (González et al., 2010), PALP heifers had a response of lower magnitude from rectal palpation, and BXKM heifers showed no increase in salivary cortisol at the 1- and 2-h sampling times, corresponding to the time the BXK would exert a clinical effect (Abrahamsen, 2013). The small rise in cortisol that appeared at the 4-h sampling period suggests the effects of the BXK had worn off by that time. It is unknown how much the analgesic or sedative effects of the BXK contributed to modulating the cortisol response.

Figure 2.

Figure 2.

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Least squares means for salivary cortisol concentrations in yearling beef heifers palpated (PALP; blue line), ovariectomized without analgesia (SPAY; red line), and ovariectomized with analgesia (BXKM; green line) on day 0, at 0 (time of palpation/ovariectomy), 1, 2, 4, 24, 48, 96, and 168 h after palpation/ovariectomy. BXKM analgesia consisted of BXK (butorphanol 0.01 mg/kg, xylazine 0.02 mg/kg, and ketamine 0.04 mg/kg) injected i.m. 5 min before ovariectomy and meloxicam (1 mg/kg) administered orally at the time of ovariectomy. Differing letters over sampling time points indicate differences between groups (P ≤ 0.05).

There were no differences (P = 0.90) in hair cortisol concentrations between treatment groups after 29 d (Table 4). Hair cortisol has been used to measure long-term cortisol secretion in several species including cattle because it accumulates in the hair and is considered a measure of the magnitude of long-term stress for up to 28 d (Moya et al., 2013). Mean hair cortisol concentrations from beef calves tended to be greater in unmedicated castrated calves (3.4 ± 0.10 nmol/L) than in calves castrated with the local anesthetic lidocaine (3.0 ± 0.10 nmol/L) 28 d after castration (Meléndez, 2018). The lack of hair cortisol increase in the study heifers suggests that the acute noxiousness of DOT ovariectomy resolved without creating long-term stress or pain.

Substance P

No treatment (P = 0.87), or treatment × time interaction (P = 0.93) effects, were detected between groups for SP (Table 4). Substance P is a neuropeptide that functions in neurotransmission, neuromodulation, inflammation, and sleep, but most interest has focused on its CNS role in nociceptive signal transmission (Mitchell et al., 2013). Its use as a pain biomarker has yielded conflicting results, such as studies that reported greater SP concentrations in untreated dehorned calves compared with those dehorned with meloxicam treatment (Coetzee et al., 2012), and greater SP concentrations in castrated calves compared with uncastrated controls (Coetzee et al., 2008), and yet another that reported no difference in SP concentrations between sham, surgical, and band castrated calves (Marti et al., 2017). Substance P may not be a useful pain biomarker for DOT ovariectomy, as there were no differences between groups detected in our study, or the pain did not reach the threshold necessary for SP release.

Acute phase proteins

Concentrations of Hp showed a treatment (P = 0.02) and treatment × time interaction effect (P < 0.001), as SPAY heifers had greater Hp concentrations than both PALP and BXKM heifers (Table 4; Fig. 3). The SPAY heifers had greater Hp concentrations than BXKM heifers at all sampling times after ovariectomy on days 1, 2, 4, and 7 (P = 0.03, P = 0.01, P < 0.001, and P = 0.01, respectively). The SPAY heifers had greater Hp concentrations than PALP heifers on day 1 (P < 0.001) and tended to have greater concentrations than PALP heifers on day 2 (P = 0.08) and day 7 (P = 0.08). The PALP heifers had a tendency to have lower Hp concentrations than BXKM heifers on day 1 (P = 0.10) but had greater concentrations than BXKM heifers on day 4 (P = 0.001). Measuring Hp and SAA provides more sensitive detection of inflammation magnitude than the CBC (Kirbas et al., 2015). Petherick et al. (2013) reported greater Hp concentrations at 0, 24 and 96 h in Bos indicus heifers that were DOT spayed without analgesia compared with unspayed controls. The Hp concentration responses in our study suggest that the heifers spayed without analgesia developed the most inflammation that had not fully resolved by day 7. Oral meloxicam has a reported half-life of 28 h (range 20 to 43 h; Coetzee et al., 2009); the lower Hp concentrations of the BXKM heifers on day 4 suggest that meloxicam provided an effective anti-inflammatory effect for DOT ovariectomy.

Figure 3.

Figure 3.

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Least squares means for the acute phase proteins serum amyloid A (top) and haptoglobin (bottom) concentrations in yearling beef heifers after palpation (PALP; blue line), ovariectomy without analgesia (SPAY; red line) and ovariectomy with analgesia (BXKM; green line) on day 0, at 0 (time of palpation/ovariectomy), 1, 2, 4, 24, 48, 96, and 168 h after palpation/ovariectomy. BXKM analgesia consisted of BXK (butorphanol 0.01 mg/kg, xylazine 0.02 mg/kg, and ketamine 0.04 mg/kg) injected i.m. 5 min before ovariectomy and meloxicam (1 mg/kg) administered orally at the time of ovariectomy. Differing letters over sampling time points indicate differences between groups (P ≤ 0.05).

The SAA concentration analysis showed a treatment × time interaction effect, as both SPAY and BXKM heifers had greater SAA concentrations than PALP heifers (P = 0.02; Table 4). At the time of spaying (day 0), there was a tendency for BXKM heifers to have greater (P = 0.06) SAA concentrations than PALP heifers, and at 1 h after ovariectomy the BXKM heifers had greater SAA concentrations than PALP heifers (P = 0.04; Fig. 3). The SPAY heifers tended toward greater SAA concentrations at 4 h post-spaying than BXKM heifers (P = 0.08). The day after ovariectomy (day 1), PALP heifers had lower SAA concentrations than SPAY (P = 0.05) and BXKM heifers (P = 0.02). A tendency for PALP heifers to have lower concentrations than SPAY heifers was observed on day 4 (P = 0.06) and day 7 (P = 0.10). Baseline concentration of SAA in healthy mature cattle was reported as 1.3 ± 0.4 mg/L (Ceciliani et al., 2012), but all groups in our study had mean concentrations greater than 50 µg/mL (mg/L) at 0, 24, 48, 96 and 168 h sampling times, which may have resulted from the handling stress associated with sampling and perhaps the juvenile age of the heifers. Mean baseline concentrations of SAA were reported as approximately 50 mg/L and rose to 122 to 153 ± 0.09 mg/L at 24 h in castrated beef calves (Meléndez, 2018); it is possible that normal baseline concentrations for peri-pubertal beef cattle under typical handling conditions are greater than for mature cattle. Although an increase in SAA occurred at 1 h in all groups, the BXKM heifers had greater concentrations; sheep were reported to have greater SAA concentrations after injection of vaccine (Ceciliani et al., 2012) but there are no reports of a rise in SAA after injection of analgesics. Some extrahepatic production of SAA occurs in tissues including the uterus; xylazine increases uterine tone (Abrahamsen, 2013) and may also have affected the SAA concentration in the BXKM heifers. The SAA concentration response at 24 h in both BXKM and SPAY heifers may reflect the acute inflammation of the ovariectomy procedure. By the 4-h sampling time BXKM heifers tended to have lower SAA concentrations than SPAY heifers, which supports an anti-inflammatory treatment effect.

Rectal temperature

A treatment × time effect between groups was detected for rectal temperature (RT; P < 0.001; Table 4). At the time of spaying (day 0), SPAY heifers tended to have higher RT than BXKM heifers (P = 0.06; Fig. 4). The BXKM heifers displayed higher RT than both PALP and SPAY heifers at 1 h (P = 0.003) and 2 h (P = 0.013 and P = 0.005, respectively) after ovariectomy. At 24 h after ovariectomy, PALP heifers had higher RT than BXKM heifers (P = 0.02) and SPAY heifers (P = 0.04). Normal range for rectal temperature (RT) in cattle is 36.7 to 39.1 °C, averaging 38.3 °C, and rises in response to pyrogens, exercise, inability to disperse heat, interference with thermoregulation from dehydration or when pharmaceuticals affect the thermoregulatory centers (Klein, 2013). The increased RT that the BXKM heifers displayed at 1 and 2 h post-ovariectomy occurred probably in response to one or more of the drugs administered. The dosage of BXK used in this study has a reported clinical effect duration of less than 2 h (Abrahamsen, 2013), so one or more of the BXK components was most likely responsible for the hyperthermia. Xylazine has been reported to cause hyperthermia by interference with thermoregulation in the CNS, which may be exacerbated by peripheral vasoconstriction (Gaynor and Muir, 2015) and may have been the cause of the transient hyperthermia in the BXKM heifers.

Figure 4.

Figure 4.

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Rectal temperature in yearling beef heifers after palpation (PALP), DOT ovariectomy without analgesia (SPAY), and DOT ovariectomy with analgesia (BXKM) on day 0. BXKM analgesia consisted of BXK (butorphanol 0.01 mg/kg, xylazine 0.02 mg/kg, and ketamine 0.04 mg/kg) injected i.m. 5 min before ovariectomy and meloxicam (1 mg/kg) administered orally at the time of ovariectomy. Differing letters over sampling time points indicate differences between groups (P ≤ 0.05).

Estradiol

Estradiol (E2) concentrations collected on day 29 did not differ between treatments (X2 = 1.9(2); P = 0.40; Table 4). The heifers in this trial were in good body condition and they should have been post-pubertal; however, their implant history was unknown. Although there were no statistical differences, the unspayed heifers had numerically greater E2 concentrations, and the only individuals with no detectable E2 concentrations were in the two groups that were spayed. Noseir (2003) reported peak estradiol concentrations of 6 ± 0.7 pg/mL in mature dairy cows, which is greater than the mean estradiol concentration of 1.2 ± 0.23 in the study heifers. Estradiol concentrations fluctuate during the estrus cycle, with the majority of the cycle at baseline concentrations, and in a group of 14 intact heifers, very few individuals would have had high enough concentrations to see statistical differences between treatments. Estradiol is mainly produced in ovarian follicles, but there is some production in other tissues such as the adrenal glands, so it is perhaps not unexpected that most of the ovariectomized heifers had detectable E2 concentrations. Jubb et al. (2003) examined DOT ovariectomized heifers a year later at slaughter and found 12.4% had ovarian remnants and no revascularized ovaries within the abdominal cavity were reported. Rapid revascularization within 1 mo of ovarian amputation would be necessary for follicular function to return (Terazono et al., 2012). Estradiol concentrations may not be a useful determinant of ovarian remnants or autografts.

Conclusions

Behavioral measurements provided minimal evidence that DOT ovariectomy was stressful or painful to yearling heifers, which may be because the surgical trauma is limited to visceral tissues and perhaps because cattle may be less likely to overtly display painful behavior to potential predators. However, DOT ovariectomy provoked acute stress, pain, and inflammation, as evidenced by increased cortisol, SAA, and HP concentrations that resolved within several days. Administering an i.m. injection of BXK 5 min before ovariectomy and oral meloxicam at the time of ovariectomy reduced the procedural stress and pain as well as the postsurgical inflammatory pain caused by the procedure. This BXK and meloxicam protocol can be used by veterinary practitioners to provide evidence-based analgesia for bovine DOT ovariectomy and may also provide a measure of chemical restraint in locations where hydraulic squeeze chutes are unavailable.

Acknowledgments

The authors gratefully recognize the exceptional help of the Agriculture and Agri-Food Canada technicians Fiona Brown and Randy Wilde, and the feedlot and beef cattle welfare staff, who helped with sample and data collection. Funding for this project was provided by the Canada/Alberta Livestock Research Trust Inc. that purchased the heifers, and the British Columbia Cattlemen’s Association (Beef Cattle Industry Development Fund; Project #520). Dr. Steven Hendrick from Coaldale Veterinary Clinic performed the palpations and ovariectomies. Pilot project heifers were generously provided by Frolek Cattle Co. Ltd., Kamloops, BC, and were ovariectomized by Dr. Jason McGillivray from Kamloops Large Animal Veterinary Clinic Ltd. Alberta Veterinary Laboratories Ltd. of Calgary, AB, Canada, supplied the Meloxicam Oral Suspension, USP used in this project.

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