• View in gallery
    Figure 1—

    Illustrations and photographs of cows that depict the locations where the MNT was serially measured in experiments 1 (A) and 2 (B) of a study conducted to evaluate IM injection of oxytetracycline for use as an experimental model to induce pain and assess the analgesic efficacy of FM in dairy cows. Prior to initiation of each experiment, the hair was removed from a 3 × 3-cm location surrounding each proposed oxytetracycline and sham injection site (at the prescapular region of the neck on both the left and right sides within a triangle delineated by the cranial edge of the scapula, spinous processes of the cervical vertebrae, and ventral aspect of the ligamentum nuchae [experiment 1 only] and over the cleft between the semimembranosus and semitendinosus muscles at the caudal aspect of the proximal third of the left and right hind limbs [experiments 1 and 2]). In experiment 1, each of 5 healthy adult nonlactating Jersey cows was administered oxytetracycline (10 mg/kg, IM), with the total volume divided equally between the shaved locations on the left hind limb and right side of the neck at hour 0. Also at hour 0, a sham injection, which involved pressing an empty syringe without a needle against the skin, was performed at the shaved locations on the right hind limb and left side of the neck. Additionally, each cow was randomly assigned to receive FM (2.2 mg/kg [0.044 mL/kg], IV; n = 3) or an equivalent volume of sterile saline (0.9% NaCl) solution (0.044 mL/kg, IV; control; 2) every 24 hours for a total of 5 treatments (ie, at 0, 24, 48, 72, and 96 hours after oxytetracycline administration). An algometer was used to measure the MNT at each shaved location before oxytetracycline administration and at 1, 6, and 24 hours after each injection of the assigned treatment. In experiment 2, each of 10 healthy adult nonlactating Holstein (n = 5) and Jersey (5) cows received oxytetracycline (10 mg/kg, IM) at the shaved location on either the left or right hind limb and a sham injection at the contralateral shaved location at hour 0. Additionally, each cow was randomly assigned to receive FM (n = 5) or sterile saline solution (5) as described for the cows of experiment 1 for a total of 10 treatments. An algometer was used to measure the MNT at each shaved location before oxytetracycline administration and at 6 and 24 hours after each injection of the assigned treatment.

  • View in gallery
    Figure 2—

    Mean ± SEM MNT over time for the oxytetracycline (white bars) and sham (control; black bars) injection sites in the neck (A) and hind limbs (B) and the mean ± SEM MNT for the oxytetracycline injection sites in the neck (C) and hind limb (D) for the 5 cows of experiment I that received FM (black bars; n = 3) or saline solution (control; white bars; 2) once daily for 5 days as described in Figure I. The algometer used to measure the MNT had a 6-mm-diameter tip and could apply increasing amounts of pressure at increments of 1.0 kgf up to a maximum of 13.61 kgf. An MNT of 13.61 kgf was recorded for all measurements that failed to elicit a behavioral response (kick, lifting of a limb, stomping of a foot, sudden shift of weight, head shake, or other voluntary movement to avoid the algometer-applied pressure). For analysis purposes, the MNT measurements for each cow were aggregated by day. *Within a day, value differs significantly (P < 0.05) from that for the control. See Figure I for remainder of key.

  • View in gallery
    Figure 3—

    Mean ± SEM MNT over time for the oxytetracycline (white bars) and sham (control; black bars) injection sites in the hind limbs (A) and mean ± SEM MNT for the oxytetracycline injection sites (B) and plasma fibrinogen concentration (C) over time for the 10 cows of experiment 2 that received FM (black bars; n = 5) or saline solution (control; white bars; 5) once daily for 10 days as described in Figure 1. The algometer used to measure the MNT in experiment 2 could apply increasing amounts of pressure at increments of 0.01 kgf up to a maximum of 45.36 kgf. See Figures 1 and 2 for remainder of key.

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Evaluation of intramuscular injection of oxytetracycline for use as an experimental model to induce pain and assess the efficacy of pain mitigation strategies in dairy cows

Alex L. Ohlheiser1Department of Animal Sciences, College of Agricultural Sciences, Colorado State University, Fort Collins, CO 80523.

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Jason K. Ahola1Department of Animal Sciences, College of Agricultural Sciences, Colorado State University, Fort Collins, CO 80523.

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Faith S. Baier1Department of Animal Sciences, College of Agricultural Sciences, Colorado State University, Fort Collins, CO 80523.

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Robert J. Callan2Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523.

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Andrea S. Lear2Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523.

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Stacey R. Byers2Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523.

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Lily N. Edwards-Callaway1Department of Animal Sciences, College of Agricultural Sciences, Colorado State University, Fort Collins, CO 80523.

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Abstract

OBJECTIVE

To evaluate IM injection of oxytetracycline as an experimental model to induce pain and assess the analgesic efficacy of flunixin meglumine (FM) in dairy cows.

ANIMALS

15 healthy nonlactating Jersey (n = 10) and Holstein (5) cows.

PROCEDURES

In the first of 2 experiments, 5 Jerseys were administered oxytetracycline (10 mg/kg, IM), divided between the right side of the neck and left hind limb. The left side of the neck and right hind limb received sham injections. Cows were also randomly assigned to receive FM (2.2 mg/kg, IV; n = 3) or an equal volume of saline (0.9% NaCl) solution (0.044 mL/kg, IV; control; 2) once daily for 5 days. The mechanical nociceptive threshold (MNT) was measured before oxytetracycline administration and at predetermined times after each injection of the assigned treatment. Experiment 2 was similar to experiment 1 except it involved 5 Jerseys and 5 Holsteins, oxytetracycline was injected only in a hind limb, and the assigned treatment was administered for 10 days.

RESULTS

For both experiments, mean MNT for the oxytetracycline injection site was consistently less than that for the sham injection site in the hind limbs, and mean MNT at the hind limb oxytetracycline injection site for FM-treated cows was greater than that for control cows beginning on day 3.

CONCLUSIONS AND CLINICAL RELEVANCE

IM injection of oxytetracycline in a hind limb reliably induced signs of pain in dairy cows and, with validation, might be useful as an experimental model for assessing pain mitigation strategies in cattle.

Abstract

OBJECTIVE

To evaluate IM injection of oxytetracycline as an experimental model to induce pain and assess the analgesic efficacy of flunixin meglumine (FM) in dairy cows.

ANIMALS

15 healthy nonlactating Jersey (n = 10) and Holstein (5) cows.

PROCEDURES

In the first of 2 experiments, 5 Jerseys were administered oxytetracycline (10 mg/kg, IM), divided between the right side of the neck and left hind limb. The left side of the neck and right hind limb received sham injections. Cows were also randomly assigned to receive FM (2.2 mg/kg, IV; n = 3) or an equal volume of saline (0.9% NaCl) solution (0.044 mL/kg, IV; control; 2) once daily for 5 days. The mechanical nociceptive threshold (MNT) was measured before oxytetracycline administration and at predetermined times after each injection of the assigned treatment. Experiment 2 was similar to experiment 1 except it involved 5 Jerseys and 5 Holsteins, oxytetracycline was injected only in a hind limb, and the assigned treatment was administered for 10 days.

RESULTS

For both experiments, mean MNT for the oxytetracycline injection site was consistently less than that for the sham injection site in the hind limbs, and mean MNT at the hind limb oxytetracycline injection site for FM-treated cows was greater than that for control cows beginning on day 3.

CONCLUSIONS AND CLINICAL RELEVANCE

IM injection of oxytetracycline in a hind limb reliably induced signs of pain in dairy cows and, with validation, might be useful as an experimental model for assessing pain mitigation strategies in cattle.

Interest in the ethical and humane treatment, production, and management of food animals is becoming increasingly important to consumers.1 The public has called for the development of pain mitigation practices for livestock.2 Although pain management is considered a high priority for animal well-being, research on the topic for cattle is lacking when compared with the volume of research available for other species, such as rodents, companion animals, and humans.3 The reason for that, in part, may be the absence of consistent, reliable, and sensitive methods to accurately recognize and measure pain in cattle.3 In the United States, FM is the only drug approved by the FDA for the treatment of pain in cattle, specifically pain associated with interdigital phlegmon (foot rot). For a drug to receive FDA approval for pain relief in a particular species, there must be a validated method for assessment of nociception in that species.4 The ability to quantitatively assess pain is an important aspect for improving the welfare of livestock species.5 Because of the variable nature of both chronic and acute pain, experimental induction of pain is believed to be a robust approach for validating pain measurement and assessing the efficacy of pain mitigation strategies.6

In veterinary research, studies that evaluate pain in conscious animals are designated as behavioral studies because all responses are considered a measure of nociception.7 Experimental models that assess nociception consist of an input-output system in which a stimulus (input) is applied and the reaction (output) by the animal is measured.7 Experimental studies of acute pain require appropriate stimuli to provoke the intended response; such stimuli are generally provided by electrical, thermal, mechanical, or chemical means. For a behavioral (conscious animals) study of nociception (ie, pain) to be effective, it must be characterized by specificity, sensitivity, validity, reliability, and reproducibility.7 With a well-designed experimental model, researchers can measure the effect of a nociceptive stimulus as well as any decrease in nociception by use of pain mitigation strategies and thereby validate the presence of nociception and the efficacy of analgesia.

Algometers can be used to apply pressure to a defined area and have been used to quantitatively assess mechanical nociception in conscious animals. The MNT is defined as the amount of pressure necessary to evoke a behavioral response indicative of pain.8 Mechanical nociceptive thresholds can be used to measure pain as well as to assess the efficacy of analgesic interventions.5 In cattle, pressure algometry has been used to assess pain associated with lameness localized to the feet9–11 and various disbudding12 and dehorning protocols in calves.13

The purpose of the study reported here was to evaluate IM injection of oxytetracycline as an experimental model to induce pain and assess the efficacy of pain mitigation strategies in cattle. Such a model is necessary to facilitate FDA approval of prospective analgesics for the treatment of pain in cattle. We chose to use oxytetracycline for pain induction because SC and IM injection of the drug to cattle causes tissue injury and swelling at the injection site and injection site lesions that persist for > 28 days after injection.14 Oxytetracycline is a broad-spectrum antimicrobial that is approved by the FDA to treat pneumonia and bovine respiratory disease complex (shipping fever), keratoconjunctivitis, foot rot, bacterial enteritis, Actinobacillosis lignieresii infections (wooden tongue), leptospirosis, wound infections, and acute metritis in beef and nonlactating dairy cattle.14 We used FM as the experimental analgesic for the study because it is the only drug approved by the FDA for the treatment of pain in cattle. Our hypotheses were that injection of oxytetracycline to cattle would cause pain at the injection site, which could be objectively measured with an algometer, and that systemic administration of FM would mitigate nociception (as measured by use of an algometer) at the oxytetracycline injection site.

Materials and Methods

All study procedures were reviewed and approved by the Colorado State University Institutional Animal Care and Use Committee (protocol No. 11-2583A). The study consisted of 2 experiments.

Experiment 1

Animals—Five healthy, adult, well-conditioned (BCS, 2.5 to 3.5 on a scale of 1 to 5, where 1 is emaciated and 5 is morbidly obese15), nonlactating Jersey cows (mean ± SD body weight, 514.4 ± 100.6 kg) were acquired by the university and maintained at the veterinary teaching hospital during the experiment to facilitate data collection. The cows were housed together in an outdoor lot, with ad libitum access to grass hay and water throughout the 120-hour (5-day) experimental period. The outdoor temperature ranged from −4°C to 18°C during the experimental period. All data were collected in a temperature-controlled facility that was maintained at 21°C. The facility was equipped with a manual head gate and chute system (without a body squeeze option) so that the cows could be safely restrained and handled during experimental procedures.

Experimental procedures—Oxytetracycline administration was designated as hour 0. At −1 hours, each cow was restrained in a standing position in the head gate and chute, and the hair was removed with a razor blade appropriate for surgical preparation of skin from a 3 × 3-cm area at each of 4 locations surrounding the proposed oxytetracycline and sham injection sites (the cleft between the semimembranosus and semitendinosus muscles at the caudal aspect of the proximal third of the left and right hind limbs and the prescapular region of the neck on both the left and right sides within a triangle delineated by the cranial edge of the scapula, spinous processes of the cervical vertebrae, and ventral aspect of the ligamentum nuchae; Figure 1). These areas were used to identify the locations for algometer placement for serial measurement of the MNT.

Figure 1—
Figure 1—

Illustrations and photographs of cows that depict the locations where the MNT was serially measured in experiments 1 (A) and 2 (B) of a study conducted to evaluate IM injection of oxytetracycline for use as an experimental model to induce pain and assess the analgesic efficacy of FM in dairy cows. Prior to initiation of each experiment, the hair was removed from a 3 × 3-cm location surrounding each proposed oxytetracycline and sham injection site (at the prescapular region of the neck on both the left and right sides within a triangle delineated by the cranial edge of the scapula, spinous processes of the cervical vertebrae, and ventral aspect of the ligamentum nuchae [experiment 1 only] and over the cleft between the semimembranosus and semitendinosus muscles at the caudal aspect of the proximal third of the left and right hind limbs [experiments 1 and 2]). In experiment 1, each of 5 healthy adult nonlactating Jersey cows was administered oxytetracycline (10 mg/kg, IM), with the total volume divided equally between the shaved locations on the left hind limb and right side of the neck at hour 0. Also at hour 0, a sham injection, which involved pressing an empty syringe without a needle against the skin, was performed at the shaved locations on the right hind limb and left side of the neck. Additionally, each cow was randomly assigned to receive FM (2.2 mg/kg [0.044 mL/kg], IV; n = 3) or an equivalent volume of sterile saline (0.9% NaCl) solution (0.044 mL/kg, IV; control; 2) every 24 hours for a total of 5 treatments (ie, at 0, 24, 48, 72, and 96 hours after oxytetracycline administration). An algometer was used to measure the MNT at each shaved location before oxytetracycline administration and at 1, 6, and 24 hours after each injection of the assigned treatment. In experiment 2, each of 10 healthy adult nonlactating Holstein (n = 5) and Jersey (5) cows received oxytetracycline (10 mg/kg, IM) at the shaved location on either the left or right hind limb and a sham injection at the contralateral shaved location at hour 0. Additionally, each cow was randomly assigned to receive FM (n = 5) or sterile saline solution (5) as described for the cows of experiment 1 for a total of 10 treatments. An algometer was used to measure the MNT at each shaved location before oxytetracycline administration and at 6 and 24 hours after each injection of the assigned treatment.

Citation: American Journal of Veterinary Research 81, 6; 10.2460/ajvr.81.6.471

At hour 0, each cow received oxytetracyclinea (10 mg/kg, IM), with the total volume divided equally between the 2 previously described injection sites in the left hind limb and right side of the neck as well as sham injections at the 2 previously described injection sites in the right hind limb and left side of the neck. The sham injections involved pressing an empty syringe without a needle against the skin at the designated sites.

Additionally, each cow was randomly assigned to receive FMb (2.2 mg/kg [0.044 mL/kg], IV; n = 3) or an equivalent volume of sterile saline (0.9% NaCl) solution (0.044 mL/kg, IV; control; 2) every 24 hours for a total of 5 treatments (ie, at 0, 24, 48, 72, and 96 hours after oxytetracycline administration). The randomization method used consisted of blindly drawing a marker from a container for each cow; the container initially held 3 markers designated for the FM treatment and 2 markers designated for saline treatment. The BCS of each cow was not considered during the random assignment of cows to the FM or saline treatment groups. The assigned treatment was injected into a jugular vein with a 16-gauge, 3.81-cm hypodermic needle while the cow was restrained in the head gate with its head further immobilized with a halter. The jugular vein used for treatment administration was alternated on a daily basis.

Study personnel responsible for administering injections and performing algometric measurements remained unaware of (were blinded to) the treatment group assignment for all cows throughout the experimental period. The assigned treatments were drawn up into syringes that were labeled with the appropriate cow's identification and given to the personnel who were responsible for injecting the treatments. The treatments could not be distinguished because both FM and saline solution are clear liquids. The person (ALO) responsible for performing the algometric measurements was not present during oxytetracycline administration and was blinded to which sites received the oxytetracycline and sham injections.

Algometry—The MNT was determined for each of the 4 injection site locations at 0 (immediately before), 1, 6, 24, 25, 30, 48, 49, 54, 72, 73, 78, 96, 97, 102, and 120 hours after oxytetracycline administration (ie, at 1, 6, and 24 hours after each injection of the assigned treatment [FM or saline solution]). The algometerc used to measure the MNT had a 6-mm-diameter tip. It could apply increasing amounts of pressure at increments of 1.0 kgf up to a maximum of 13.61 kgf. At each designated time, the tip of the algometer was applied perpendicularly to the center of each 3 × 3-cm shaved area. Pressure was applied to the site at a rate of approximately 5 kgf/s until a behavioral response was elicited or a maximum pressure (13.61 kgf) was achieved. A behavioral response was defined as a kick, lifting of a limb, stomping of a foot, sudden shift of weight, head shake, or other voluntary movement to avoid the algometer-applied pressure. The pressure applied at the time of the behavioral response was recorded as the MNT. An MNT of 13.61 kgf was recorded for all measurements that failed to elicit a behavioral response. The MNT was determined in triplicate at each location with at least a 1-minute interval between successive measurements, and the mean for the 3 measurements was calculated and used for analysis purposes. At each designated time, the order in which the MNT was determined for the 4 locations was randomized by means of blindly drawing markers from a container. The markers were labeled right or left and indicated which oxytetracycline injection site (left hind limb or right neck) would be measured first. The algometer was applied alternatively between the hind limbs or sides of the neck until all 3 MNT measurements were obtained, then the process was repeated for the 2 remaining injection sites. When multiple study procedures were scheduled for a designated time, measurement of the MNT was always performed first.

Experiment 2

Animals—The number of cows necessary for experiment 2 was determined on the basis of results of a power calculation as described,16 which used the results from experiment 1 including the observed means and SEM for the MNT. Ten healthy, adult, well-conditioned (BCS range, 2.5 to 3.5), nonlactating Jersey (mean ± SD body weight, 478.4 ± 50.1 kg; n = 5) and Holstein (mean ± SD body weight, 689.0 ± 15.4 kg; 5) cows that had been culled from their herds of origin were acquired by the university. The cows were culled because of poor milk production or reproductive performance and not because of health-or pain-related issues. The cows were maintained at the veterinary teaching hospital as described for the cows of experiment 1 for the duration of the 240-hour (10-day) experimental period. The outdoor temperature ranged from −4°C to 18°C during the experimental period, and all data collection took place in a temperature-controlled facility that was maintained at 21°C.

Experimental procedures—Oxytetracycline administration was designated as hour 0. Prior to oxytetracycline administration, each cow was restrained in the head gate and chute and hair was removed from a 3 × 3-cm area at each of 2 locations (caudal aspect of the proximal third of the left and right hind limbs over the semimembranosus and semitendinosus muscles) as described for experiment 1 (Figure 1).

A blood sample (10 mL) was collected by jugular venipuncture with a 16-gauge, 3.81-cm needle into a sterile 10-mL syringe at 0 (immediately before; baseline), 24, 48, 72, 96, 120, 144, 168, 192, 216, and 240 hours after oxytetracycline administration for determination of plasma fibrinogen concentration. Each blood sample was transferred to a sterile blood collection tube that contained EDTA as an anticoagulant immediately after collection. The tube was gently inverted several times to ensure that the blood was adequately mixed with the EDTA to prevent coagulation. For each sample, the plasma fibrinogen concentration was determined within 2 hours after collection by means of a heat precipitation method as described.17,18

Within each breed, cows were randomly assigned as described for experiment 1 to receive FM (2.2 mg/kg, IV; n = 5) or an equivalent volume of sterile saline solution (0.044 mL/kg, IV; control; 5) every 24 hours for a total of 10 treatments (ie, at 0, 24, 48, 72, 96, 120, 144, 168, 192, and 216 hours after oxytetracycline administration). The randomization method ensured that the breed distribution did not differ significantly between the 2 treatment groups. The assigned treatment was administered into a jugular vein as described for experiment 1.

At hour 0, each cow received oxytetracycline (10 mg/kg, IM). The drug was injected in the middle of the shaved area on the caudal aspect of the proximal third of the right or left hind limb. The leg in which the oxytetracycline was injected was determined randomly by blindly drawing a marker from a container. A sham injection was performed in the contralateral leg. All injections were performed as described for experiment 1. Administration of the oxytetracycline and sham injections was the last study procedure performed at hour 0 (ie, was performed after MNT measurement, blood sample collection, and administration of the assigned treatment [FM or saline solution]).

As in experiment 1, all study personnel responsible for administering injections and performing algometric measurements were blinded to the treatment group assignment for all cows throughout the experimental period. Also, the person (ALO) responsible for performing the algometric measurements was blinded to which sites received the oxytetracycline and sham injections. The observation period for experiment 2 was extended to 10 days so that the effects of the pain stimulus and analgesia could be observed for a longer duration than in experiment 1 (5 days).

Algometry—For each cow, the MNT was determined for each shaved location at 0 (immediately before; baseline), 6, 24, 30, 48, 54, 72, 78, 96, 102, 120, 126, 144, 150, 168, 174, 192, 198, 216, 222, and 240 hours after oxytetracycline administration (ie, immediately before and at 6 hours after each injection of the assigned treatment) as described for experiment 1. The algometerc used to measure the MNT in experiment 2 was similar to that used in experiment 1 except it could apply increasing amounts of pressure at increments of 0.01 kgf up to a maximum of 45.36 kgf. An MNT of 45.36 kgf was recorded for all measurements that failed to elicit a behavioral response. When multiple procedures were performed at a designated time, the MNT was measured first, followed by blood sample collection and administration of the assigned treatment.

Statistical analysis

All analyses were performed with commercially available statistical software,d and values of P < 0.05 were considered significant. Cow was the experimental unit for all analyses. The effect of various covariates on MNT was assessed by means of an ANOVA for repeated measures. For experiment 1, the model included fixed effects for treatment (FM or saline solution), site (right or left neck or right or left hind limb), MNT acquisition time (time), and BCS and a random effect to account for repeated measurement of the MNT within each cow. The model for experiment 2 was similar to that for experiment 1 with the addition of fixed effects for breed (Holstein or Jersey) and the interaction between breed and site. The Kenward-Roger method was used to determine denominator degrees of freedom. Initially, the models for both experiments included a fixed effect for baseline MNT (ie, the MNT measured prior to injection of the assigned treatment and oxytetracycline); however, it did not have a significant (P = 0.41) effect on subsequent MNT measurements and was removed from both models. The same was true for BCS. Although MNT was significantly affected by time (P < 0.05), it was not significantly associated with the interaction between time and site (P = 0.81), the interaction between time and treatment (P = 0.26), or the 3-way interaction for time, site, and treatment (P = 0.66). Because the main effects of the model did not change dependent on time, the MNT was aggregated by day for comparison purposes.

Results

Experiment 1

For the 5 cows of experiment 1, the mean MNT did not differ significantly between the oxytetracycline and sham injection sites in the neck at any time during the 5-day experimental period (Figure 2) Similarly, the mean MNT at the oxytetracycline injection site in the neck did not differ significantly between the cows that received FM and those that received saline solution (controls) at any time during the experimental period. A total of 160 MNT measurements (16 measurements × 2 sites × 5 cows) were obtained in the neck region for the cows of experiment 1, and the maximum MNT (13.61 kgf) was recorded for 25 (15.6%) of those measurements.

Figure 2—
Figure 2—

Mean ± SEM MNT over time for the oxytetracycline (white bars) and sham (control; black bars) injection sites in the neck (A) and hind limbs (B) and the mean ± SEM MNT for the oxytetracycline injection sites in the neck (C) and hind limb (D) for the 5 cows of experiment I that received FM (black bars; n = 3) or saline solution (control; white bars; 2) once daily for 5 days as described in Figure I. The algometer used to measure the MNT had a 6-mm-diameter tip and could apply increasing amounts of pressure at increments of 1.0 kgf up to a maximum of 13.61 kgf. An MNT of 13.61 kgf was recorded for all measurements that failed to elicit a behavioral response (kick, lifting of a limb, stomping of a foot, sudden shift of weight, head shake, or other voluntary movement to avoid the algometer-applied pressure). For analysis purposes, the MNT measurements for each cow were aggregated by day. *Within a day, value differs significantly (P < 0.05) from that for the control. See Figure I for remainder of key.

Citation: American Journal of Veterinary Research 81, 6; 10.2460/ajvr.81.6.471

For the hind limbs, the mean MNT for the oxytetracycline injection site did not differ significantly (P = 0.23) from that for the sham injection site at baseline (day 0) but was significantly (P ≤ 0.05) lower than the mean MNT for the sham injection site (ie, the oxytetracycline injection site was more sensitive to applied pressure) on the subsequent 5 days of the experimental period (Figure 2). The mean MNT for the oxytetracycline injection site in the hind limb did not differ significantly between the FM-treated and control cows on days 0, 1, and 2. However, the mean MNT at the oxytetracycline injection site in the hind limb for the FM-treated cows was significantly greater than that for the control cows (ie, the oxytetracycline injection site was less sensitive for FM-treated cows, compared with that for the control cows) on days 3, 4, and 5. A total of 160 MNT measurements were obtained in the hind limb region for the cows of experiment 1, and the maximum MNT was recorded for 32 (20%) of those measurements.

Experiment 2

For the 10 cows of experiment 2, the mean MNT did not differ significantly between the oxytetracycline and sham injection sites at baseline. However, the mean MNT at the oxytetracycline injection site was significantly less than that at the sham injection site on each of the successive 10 days (Figure 3) Compared with the MNT at the oxytetracycline injection site for the control cows, the mean MNT at the oxytetracycline injection site for the FM-treated cows did not differ significantly at baseline or days 1 and 2 but was significantly greater on days 3, 4, 6, 7, and 9. The mean MNT at the oxytetracycline injection site for the FM-treated cows was numerically greater than that for the controls on days 5, 8, and 10, but did not differ significantly between groups on any of those 3 days. During experiment 2, the maximum MNT (45.36 kgf) was not achieved during any algometric measurement.

Figure 3—
Figure 3—

Mean ± SEM MNT over time for the oxytetracycline (white bars) and sham (control; black bars) injection sites in the hind limbs (A) and mean ± SEM MNT for the oxytetracycline injection sites (B) and plasma fibrinogen concentration (C) over time for the 10 cows of experiment 2 that received FM (black bars; n = 5) or saline solution (control; white bars; 5) once daily for 10 days as described in Figure 1. The algometer used to measure the MNT in experiment 2 could apply increasing amounts of pressure at increments of 0.01 kgf up to a maximum of 45.36 kgf. See Figures 1 and 2 for remainder of key.

Citation: American Journal of Veterinary Research 81, 6; 10.2460/ajvr.81.6.471

The mean plasma fibrinogen concentration did not differ between the FM-treated and control cows at baseline prior to administration of oxytetracycline and the initial dose of the assigned treatment (Figure 3). However, the mean plasma fibrinogen concentration for the FM-treated cows was significantly lower than that for the control cows on days 5 and 6.

Discussion

The primary objective of the present study was to develop and evaluate an experimental model to induce pain in cattle that could be used to objectively assess the efficacy of pain mitigation strategies. Results of this study suggested that IM injection of oxytetracycline in the caudal aspect of the semimembranous and semitendinosus muscles at the proximal third of a hind limb could be used to induce signs of pain in cattle. In experiment 1 of this study, oxytetracycline was injected in the right side of the neck (prescapular region within a triangle delineated by the cranial edge of the scapula, spinous processes of the cervical vertebrae, and ventral aspect of the ligamentum nuchae) as well as the caudal aspect of the proximal third of the left hind limb. Sham injections were performed in the left side of the neck and right hind limb. The mean MNT did not differ significantly between the oxytetracycline and sham injection sites in the neck but was significantly lower for the oxytetracycline injection site, compared with the sham injection site in the hind limbs. This suggested that the neck was not a good location for objective assessment of MNT in cattle. A measurable change in response to a painful stimulus is crucial for evaluation of the efficacy of a pain mitigation strategy.19 Moreover, assessment of pain in prey animals such as cattle is challenging because those animals tend to instinctively conceal signs of pain.20 The cows of experiment 1 were more aware of the researcher responsible for acquiring MNT data when measurements were obtained in the neck region than when measurements were obtained in the hind limbs. Also, the application of the algometer at the neck locations required restraint of the head and neck, and cows tend to inherently resist manipulation of the head and neck. A similar fear-induced inhibition of behavioral responses was observed in ewes when an algometer was used to measure the MNT of forelimbs.5

In experiment 1 of the present study, oxytetracycline was injected in the right side of the neck and left hind limb, which could have confounded the results if nociceptive processing in cattle is associated with 1 hemisphere of the brain as it appears to be in humans.21 That might have contributed to the discrepancy in behavioral responses when the algometer was applied to the neck versus the hind limbs. Other factors that might have contributed to the differences in the MNT measurements obtained in the neck region versus those measured in the hind limbs include the fairly small sample size (n = 5) and restraint of the cows in a head gate and chute, which limited head and neck movement to a much greater extent than hind limb movement. Although it is possible that, for the cows of experiment 1, injection of oxytetracycline at 2 sites might have caused hyperalgesia (increased pain sensitivity [ie, a decrease in MNT]) that could have affected the MNT, we do not believe that hyperalgesia substantially contributed to the overall behavioral responses of the cows to the algometer. An algometer has been successfully used to measure the MNT at the horn region of calves following various disbudding12 and dehorning13 procedures. Nonetheless, because the neck appeared to be an ineffective location for measurement of the MNT for the cows of experiment 1, the MNT was measured only in the hind limbs for the cows of experiment 2.

In both experiments 1 and 2 of the present study, the mean MNT did not differ between the oxytetracycline and sham injection sites in the hind limbs at baseline (ie, before oxytetracycline administration) but was consistently lower for the oxytetracycline injection site relative to the sham injection site for the remainder of the experimental period. That finding suggested IM injection of oxytetracycline in the hind limbs of cattle was an effective chemical stimulus to induce pain and enhance the MNT at the injection site. For an experimental model of nociception to be valid, the nociceptive response to a stimulus must be predictable, repeatable, and measurable.7 Results of experiments 1 and 2 of the present study indicated that IM injection of oxytetracycline to cattle caused an inflammatory response and signs of pain at the injection site that were predictable and repeatable. Approximately half the cows in both experiments 1 and 2 received the NSAID FM. For those cows, it is possible that the inflammation induced by IM injection of oxytetracycline was mitigated by the anti-inflammatory (and analgesic) properties of FM, which may have confounded comparison of the MNT between the oxytetracycline and sham injections sites within individual cows (ie, resulted in no significant differences between the 2 injection sites). However, following oxytetracycline administration, the MNT for the oxytetracycline injection site was consistently lower than that for the sham injection site for all cows of both experiments. Further research is necessary to validate IM injection of oxytetracycline for the experimental induction of pain in cattle.

A secondary hypothesis of the present study was that the nociceptive response of cattle following IM injection of oxytetracycline would be mitigated by administration of FM. The extent of that mitigation could be objectively measured with an algometer. An increase in MNT is indicative of a loss of sensation or increase in tolerance to the applied stimulus at the evaluated location.22 The mean MNT for the oxytetracycline injection site in the hind limb for FM-treated cows was significantly greater than that for the control cows on days 3, 4, and 5 of experiment 1 and days 3, 4, 6, 7, and 9 of experiment 2. This suggested that FM effectively mitigated the pain induced by oxytetracycline at the injection site. Similarly, sows with experimentally induced lameness had a significantly greater MNT following administration of FM or meloxicam (another NSAID), compared with similar sows that received a saline solution placebo.23

The nociceptive benefits of FM did not become significantly evident until day 3 in both experiments 1 and 2 of the present study. The reason for that was unknown. The mean MNT at the hind limb oxytetracycline injection site for FM-treated cows was numerically greater than that for the control cows on days 1 and 2 in both experiments. It is possible that there was insufficient power to detect a significant difference between the 2 groups of cows owing to the small sample size in both experiments. Alternatively, the effect of FM on the MNT may have been additive such that the MNT increased as the duration of treatment progressed. In experiment 2, the mean MNT at the oxytetracycline injection site did not differ significantly between the FM-treated and control cows on days 5, 8, and 10. The apparent lack of consistency in the MNT comparison between FM-treated and control cows on successive days was likely caused by varying pain tolerance in individual cows or varying response of cows to the presence of the researcher or the pressure applied by the algometer. Also, the small number of cows limited the power to overcome the inherent variation in behavioral responses among cows.

For each experiment of the present study, the ANOVA model built to assess MNT included a random effect to account for variability among repeated measures within individual cows. Within-animal variability in MNT was observed for sheep of another study.5 In the present study, differences in the physical size of the cows were controlled by administration of both oxytetracycline and the assigned treatment on a mg/kg or mL/kg basis. Body condition score was not significantly associated with MNT and was therefore excluded from the final model for both experiments. The remaining variation in the models was most likely attributable to individual cow temperament, sensitivity to pain, and general nature toward handlers.

In cattle, plasma fibrinogen concentration is commonly quantified for assessment of an acute-phase inflammatory response.24,25 Local tissue inflammation causes production and release of cytokines that stimulate the production and release of acute-phase proteins, such as haptoglobin and fibrinogen from the liver.26 On days 5 and 6 of experiment 2, the mean plasma fibrinogen concentration for the FM-treated cows was significantly lower than that for the control cows, which suggested that the drug successfully decreased local inflammation and cytokine signals responsible for eliciting fibrinogen release that were induced by IM injection of oxytetracycline.

Results of the present study suggested that IM injection of oxytetracycline in the caudal aspect of the proximal third of a hind limb could be used to experimentally induce signs of pain in cattle. The amount of pain induced by that method could be objectively measured with an algometer. Use of an algometer to measure the MNT allowed us to assess the efficacy of FM for mitigation of pain at the oxytetracycline injection site. Further research is necessary to validate the use of IM injection of oxytetracycline to experimentally induce pain and use of pressure algometry to objectively and reliably assess injection site hyperalgesia in cattle. Validation of an effective method for experimental induction of pain in cattle is critical for the development of a reliable protocol to assess the efficacy of pain mitigation strategies in cattle.

Acknowledgments

No third-party funding or support was received in connection with this study or the writing or publication of the manuscript. The authors declare that there were no conflicts of interest.

ABBREVIATIONS

BCS

Body condition score

FM

Flunixin meglumine

kgf

Kilogram-force

MNT

Mechanical nociceptive threshold

Footnotes

a.

Oxytetracycline Injection 200, Norbrook Inc, Lenexa, Kan.

b.

Prevail, Vet One, Boise, Idaho.

c.

FPK, Wagner Instruments, Greenwich, Conn.

d.

SAS, version 9.4, SAS Institute Inc, Cary, NC.

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Contributor Notes

Mrs. Ohlheiser's present address is Leachman Cattle of Colorado, Fort Collins, CO 80524.

Ms. Baier's present address is the Department of Animal Sciences, College of Agricultural and Life Sciences, University of Wisconsin-Madison, Madison, WI 53715.

Dr. Lear's present address is the Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996.

Dr. Byers' present address is the Department of Large Animal Medicine and Surgery, School of Veterinary Medicine, St George's University, Grenada, West Indies.

Address correspondence to Dr. Ahola (jason.ahola@colostate.edu).