Carrageenan-induced inflammation elicits behavioral changes in cockatiels (Nymphicus hollandicus) for potential pain scale development

Nicole A. Mikoni Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California-Davis, Davis, CA

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David Sanchez-Migallon Guzman Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California-Davis, Davis, CA

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Hugues Beaufrere Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California-Davis, Davis, CA

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Joanne R. Paul-Murphy Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California-Davis, Davis, CA

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Abstract

OBJECTIVE

To evaluate behaviors associated with inflammatory pain induced by carrageenan injection in the cockatiel and determine interobserver agreement.

ANIMALS

16 adult cockatiels.

METHODS

Cockatiels were randomly assigned as either treatment (carrageenan injection) or control (sham injection) group. The treatment group received a subcutaneous injection of 0.05 mL of a 1% lambda carrageenan solution into the left footpad. Following treatment or control procedures, all cockatiels were video recorded individually for 9.5 hours. Ten minutes of video at each of 11 time points postinjection and/or handling were evaluated by 3 different observers. Twenty-five behaviors within 6 categories (resting, locomotion, maintenance, intake, interaction with environment, and limb and body posture) were assessed, in addition to crest position and mentation. Differences in individual behaviors tallies were assessed using serial Wilcoxon sum rank tests. Interobserver agreement was assessed using an intraclass correlation coefficient for a 2-way design for consistency among multiple observers.

RESULTS

Treatment cockatiels exhibited significantly increased focal preening (q = .023) and increased burst preening (q = .036), while control cockatiels spent significantly more time in an upright stance (q = .036). Although the remainder of behaviors observed were not statistically significant between groups, additional variables of interest seen more frequently in treatment cockatiels included non–weight-bearing stance, holding of the body low, and being nonvigilant. The level of agreement between observers was variable based on the specific behaviors; nevertheless, the dynamic behaviors were substantial to strong.

CLINICAL RELEVANCE

Carrageenan-induced inflammation-associated behaviors may be valuable in developing a pain scale and evaluating mild inflammatory pain in small psittacine species.

Abstract

OBJECTIVE

To evaluate behaviors associated with inflammatory pain induced by carrageenan injection in the cockatiel and determine interobserver agreement.

ANIMALS

16 adult cockatiels.

METHODS

Cockatiels were randomly assigned as either treatment (carrageenan injection) or control (sham injection) group. The treatment group received a subcutaneous injection of 0.05 mL of a 1% lambda carrageenan solution into the left footpad. Following treatment or control procedures, all cockatiels were video recorded individually for 9.5 hours. Ten minutes of video at each of 11 time points postinjection and/or handling were evaluated by 3 different observers. Twenty-five behaviors within 6 categories (resting, locomotion, maintenance, intake, interaction with environment, and limb and body posture) were assessed, in addition to crest position and mentation. Differences in individual behaviors tallies were assessed using serial Wilcoxon sum rank tests. Interobserver agreement was assessed using an intraclass correlation coefficient for a 2-way design for consistency among multiple observers.

RESULTS

Treatment cockatiels exhibited significantly increased focal preening (q = .023) and increased burst preening (q = .036), while control cockatiels spent significantly more time in an upright stance (q = .036). Although the remainder of behaviors observed were not statistically significant between groups, additional variables of interest seen more frequently in treatment cockatiels included non–weight-bearing stance, holding of the body low, and being nonvigilant. The level of agreement between observers was variable based on the specific behaviors; nevertheless, the dynamic behaviors were substantial to strong.

CLINICAL RELEVANCE

Carrageenan-induced inflammation-associated behaviors may be valuable in developing a pain scale and evaluating mild inflammatory pain in small psittacine species.

Recognition and assessment of pain allow the provision of supportive care and administration of analgesics, improving the welfare of a given patient. However, recognition and assessment of pain are still in early development for birds, creating ambiguity in the evaluation of how painful a bird is. If pain is not recognized or is underestimated, a bird might not receive appropriate treatment. Inadequately treated pain can result in increased morbidity and mortality and a poor quality of life for both the patient and the caretaker.13

The International Association for the Study of Pain describes pain as an “unpleasant sensory and emotional experience associated with, or resembling that associated with, actual or potential tissue damage.”4 This definition of pain was expanded as it pertains to animals as “an aversive sensory experience caused by actual or potential injury that elicits protecting motor and vegetative reactions, results in learned avoidance behavior, and may modify species-specific behavior, including social behavior.”5,6 An animal’s inability to verbally communicate with humans does not negate an animal’s ability to perceive and respond to painful stimuli; however, accurate recognition and assessment of the response to pain remain challenging and particularly difficult for animal subgroups comprised of a large number of diverse species.7

Pain scales have been validated in mammalian species and gained increased clinical value to maintain acceptable welfare in both research and clinical animal patients.810 These tools include observation and assessment of facial structure, body posture, activity, and interaction with others (conspecifics, owners, etc) as a means of evaluating and quantifying discomfort. Behavioral and physiologic changes that accompany painful conditions have become essential in assessing pain in domestic species, particularly dogs, cats, and rabbits. A similar application of pain scales for avian species would be highly valuable; however, challenges exist given the different behaviors of avian species and types of pain. Variations in anatomical and physiologic traits of avian species compared to mammals also limit the utilization of grimace scales and composite pain scales that rely on changes in facial expression. Familiarity with the normal, nonpainful behaviors of an avian species of interest is critical, as these can be unique to both the species and the individual. Disparities in natural behaviors between avian species have been recognized in prey and predatory species, as well as with individual rearing, particularly in terms of socially raised versus individually raised captive birds.1113 Additionally, the introduction of pain to an animal will concurrently introduce stress. Stress, even in the absence of pain, will significantly alter behavior, as documented in studies performed in European starlings (Sturnus vulgaris)14 and trumpeter swans (Cygnus buccinator).15 An observer studying pain behaviors in a bird would ideally have access to published information and be familiar with the species’ behavioral response to stress to avoid confounding behavioral alterations.

Psittacine birds are common companion animals, and cockatiels (Nymphicus hollandicus) are the most popular companion psittacine species in the US.16 Psittacine birds are frequently presented to veterinarians in private practice, zoological collections, and rehabilitation facilities for injuries and diseases in which pain recognition and subsequent pain management are needed. The cockatiel was therefore selected as an appropriate species for our initial studies on behavioral recognition of pain using an acute inflammatory pain model.17 Both normal (unstressed) and stress ethograms have been published for the cockatiel,18 which is beneficial for distinguishing between behavioral changes that accompany stressful situations (handling, change in environment, etc) and those that are more indicative of acute inflammatory pain, such as the one investigated in the model for this study.

The induction of acute inflammation and associated hyperalgesia with carrageenan is one of the most widely used and well-established rodent models for studying acute inflammation that persists for a limited period of time.19,20 Carrageenan is a sulphated mucopolysaccharide that is extracted from Irish moss (Chondrus crispus) and, when injected, stimulates local inflammatory responses primarily through the aggregation of macrophages.21 Inflammation induced by carrageenan is acute, does not stimulate the adaptive immune system, is reproducible, and has been valuable in inducing hyperalgesic conditions utilized to assess the effectiveness of certain analgesics.19,22 Carrageenan-induced inflammatory studies22,23 have been utilized to evaluate weight-bearing and behavioral changes associated with hyperalgesia in rats. Carrageenan has been applied in domestic fowl chicks causing both anatomical changes24 and differences in withdrawal latencies.25 The administration of carrageenan in small species such as rats, mice, and domestic fowl chicks suggests that administration into cockatiels, with an average weight of 100 grams, should be both feasible and reproducible.19,22,25 Basic protocols for carrageenan administration have been established,26 and the resolution of acute inflammatory changes following administration of anti-inflammatory analgesics in rodent models supports humane-use-in-a-laboratory-setting.22

The purpose of this study was to document behavioral changes associated with carrageenan-induced acute inflammatory pain of the footpad in cockatiels and to evaluate for any behavioral modifications that may be present in instances of acute inflammatory discomfort. We hypothesized that the behavioral profile of cockatiels in pain (treatment cockatiels) would be significantly different from that of cockatiels affected by stress (control cockatiels) alone. An additional hypothesis included that pain-related behaviors would be readily recognized by the observers evaluating the cockatiels in this study.

Methods

Animals

Sixteen 3-year-old normal gray color morph cockatiels (8 females and 8 males) served as the study population. An intake physical examination was performed on each cockatiel to ensure appropriate health before the study. Cockatiels used for this study were housed individually in standard wire mesh laboratory cages (30.5 X 30.5 X 30.5 cm) with a wooden perch, hanging toy, feeder trough, and water drip line. Birds were acclimated to the study housing for 14 days before study initiation to minimize confounding effects of stress and minor variations in standard lighting and temperature conditions. All study cockatiels were housed in the same room to provide visual and auditory stimuli to one another to minimize adverse behavioral effects of isolation of this social psittacine species. Cockatiels were maintained on a day/night cycle of 10 hours of light and 14 hours of darkness, fed a commercial pelleted diet (Roudybush Inc), and provided water ad libitum through the drip system. The room temperature was maintained between 22.2 °C and 22.8 °C, and the humidity was between 20% and 50%. The study was approved by the Institutional Animal Care and Use Committee at the University of California-Davis.

Normal behaviors

Recording equipment included 16 individual 3 Megapixel High Definition Bullet cameras attached to a single Network Video Recorder (NVR) Security System recording device (LOREX Technology Inc). A computer monitor (Dell Technologies) was attached to the NVR System to display the video feed from all 16 cameras on a single screen. Cameras were attached to a single metal rack using zip ties and positioned so that a single cockatiel’s cage was centered within the view of each camera. To acclimate the 16 cockatiels to the presence of the cameras, the rack of cameras was initially placed 12 feet from the cockatiel cages and then moved closer to their cages by approximately 6 inches each day. When cameras were within an ideal proximate distance of 6 to 8 inches from the cockatiel cages, the camera rack was kept in this position for the viewing and recording of each cockatiel. Recording hours (7 AM to 5 PM daily) were customized through the NVR System settings, and collected video was stored on the system’s hard drive. Recordings were downloaded daily after completion onto an external hard drive for further evaluation.

Three days before carrageenan and sham treatments, an uninterrupted 10 hours of video recording (7 AM to 5 PM) were obtained for all 16 cockatiels, and no personnel entered the room during the recording to reduce potential effects on behavior due to human presence. All videos were viewed at established time points (baseline “hour 0,” 0.5, 1, 2, 3, 4, 5, 6, 7, 8, and 9 hours from initial recording start) to determine the frequency of each cockatiel’s behaviors, crest positions, and mentation during the undisturbed laboratory setting. Husbandry staff was then allowed to enter the room to refill the feeder troughs and replace the paper lining beneath each cage as per their daily care routine.

Observation of various behaviors displayed by the undisturbed cockatiels was performed using focal continuous sampling and an “all occurrences” method. For this study, 25 different behaviors were observed and used to create the quantitative ethogram using the format of published cockatiel ethograms (Table 1).18 Additionally, the crest position (erect, lowered, or flat) and mentation (alert, quiet, depressed, or nonvigilant) of each cockatiel were monitored. The total number of each behavior was counted (cumulative tally of each time a behavior was observed) for each time point over a period of 10 minutes per time point, with a notation on the overall displayed crest position and mentation being recorded at the end of each 10-minute section. Each cockatiel was observed through video recordings for a total of 110 minutes before any allocation into treatment or control group. A description of each of the 25 different behaviors, 3 different crest positions, and 3 different mentation statuses is as follows: (1) perch resting = sitting still on perch or lip of feeder trough with eyes open and not engaging in any other active behaviors for at least 10 continuous seconds; (2) floor resting = sitting still on cage floor with eyes open and NOT engaging in any other active behaviors for at least 10 continuous seconds; (3) nonvigilant = resting in 1 place with eyes at least 50% partially closed or fully closed and/or resting with head tucked on top of back or under a wing for at least 10 continuous seconds; (4) moving along cage floor = taking 3 or more steps while running or walking along floor of cage; (5) active climbing = supporting full body weight with legs from cage wall or climbing along walls of cage; (6) moving along perch = climbing onto and moving 3 or more steps laterally across wooden perch; (7) thorough preening = thorough grooming and feather maintenance of self for 10 or more continuous seconds; (8) burst preening = short duration grooming lasting less than 5 seconds and with rapid return of focus to surroundings; (9) focal preening = focused grooming of 1 particular part of the body for 5 or more continuous seconds; (10) rouse = quick rustling or shaking of feathers to settle them back in place; (11) scratching = scratching body with foot; (12) stretching = stretching limbs (wings, legs) or body/neck; (13) wiping beak = wiping beak on limb or object in cage; (14) eating = placing beak into food trough and retrieving pieces of food in the mouth; (15) drinking = at the water line drinking water; (16) playing with/inspecting toy = engaged with hanging toy in cage corner; (17) biting on perch = biting/chewing on wooden perch; (18) biting cage bars = biting/chewing on metal cage bars (while not actively climbing); (19) interaction with food trough = pecking food trough, clacking beak against food trough, or quickly hopping on and off food trough but not consuming feed; (20) engaged with nearby conspecific = placing self near cage wall closest to conspecific and looking at/interacting with nearby conspecific or performing courtship displays; (21) upright = body straight and with both limbs placed on contact surface (floor, perch, cage wall, etc); (22) leaning = displacing weight from 1 limb but still keeping both limbs on ground; (23) non–weight-bearing = hovering or complete holding up of 1 limb; (24) requiring bodily support = displacing full body weight onto vertical cage wall surface for support; (25) holding body low on cage floor = body held low to ground with drawn-in appearance to head, neck, and body. Crest positions were as follows: erect = crest feathers are fully displayed and very easily visualized, with top crest feather being at a greater than 45° angle; lowered = crest feathers are drooped but still individually visible; and flat = crest feathers are pressed down against curvature of head and difficult to visualize outside of tips of crest feathers. Mentation was as follows: alert = both eyes open and with cockatiel observing or engaging with environment; quiet = both eyes open but with cockatiel exhibiting resting behavior for majority of time observed; and depressed = eyes squinted or closed and with cockatiel remaining in 1 position in cage for majority of time observed but NOT displaying nonvigilant behavior.

Table 1

Behaviors recorded during observation of cockatiels and utilized to evaluate for differences between control (handling only; n = 8) and treatment (carrageenan injected; 8) cockatiels.

Behavior category Behavior
Resting Perch resting
Floor resting
Nonvigilant
Locomotion Moving along cage floor
Active climbing
Moving along perch
Maintenance Thorough preening
Burst preening
Focal preening
Rouse
Scratching
Stretching
Wiping beak
Intake Eating
Drinking
Interaction with environment Playing with/inspecting toy
Biting on perch
Biting cage bars
Interaction with food trough
Engaged with nearby conspecific
Limb and body posture Upright
Leaning
Non–weight-bearing
Requiring bodily support
Holding body low on cage floor
Crest position (overall assessment after 10 minutes) Erect
Lowered
Flat
Mentation (overall assessment after 10 minutes)—cockatiel must be awake to assess Alert
Quiet
Depressed

Study groups and induction of inflammation

The carrageenan for this study was the lambda type formulated as a 1% lambda carrageenan solution (TCI America) using a deionized water vehicle and prepared sterilely as previously published.26 A single 50-mL batch of 1% lambda carrageenan solution was used for all injection sessions, with individual aliquots of 1.0 mL portioned and stored at −62.2 °C for 24 to 120 hours before use, depending on the predetermined study day. On each day of carrageenan injections, a single aliquot was removed from the freezer, thawed back to a liquid state over a period of approximately 10 minutes by being held in a warm hand, and then immediately drawn up into individual 30-unit (0.3 mL) U100 insulin syringes for injection (1 syringe used per cockatiel).

Cockatiels were separated based on their location on the cage rack (4 birds on the top row, 6 birds on the middle row, and 6 birds on the bottom row). For each row, half of the cockatiels were randomly assigned to either the treatment group (carrageenan injection) or control group (handling only), with randomization additionally balanced for sex and room location. The birds were randomly allocated to the treatment or control group using a sequence from random.org. The cockatiels were manually restrained, and the left foot was handled by the individual administering treatment (0.05 mL 1% carrageenan injection, per prior cockatiel carrageenan injection protocols)17 or sham (placement of a capped needle along the plantar aspect of the footpad) procedures. The carrageenan injections were performed using a 30-unit U100 insulin syringe with a 29-gauge needle. Cockatiels were each handled for an average of 1 to 2 minutes and returned to their cage for subsequent behavioral observation. Following completion of video recordings approximately 10 hours postinjection, the treatment cockatiels received meloxicam 2 mg/kg (IM injection with Ostilox; Vet One for the first dose followed by oral administration with Metacam; Boehringer Ingelheim for all other doses,) twice daily for 7 days.

Pain-related behaviors

Recordings following carrageenan injection study days were performed uninterrupted and void of human presence from 7 AM to 5 PM, similar to the prior undisturbed behavior recordings. A total of approximately 9.5 hours of uninterrupted video recording were obtained for each bird, and a single observer (NAM) initially reviewed these videos to ensure the applied ethogram was appropriate. A total of 3 additional individual observers were then collectively trained to recognize the various behaviors displayed in the established ethogram and to apply this ethogram to quantify behaviors observed during the study periods. All observers were undergraduate students chosen based on past participation in animal behavioral studies, previous avian experience, and education in animal-related fields. Individuals evaluating the behavioral recordings were masked to the assignment of treatment versus control animals. For each observer, the videos of each cockatiel were viewed separately, and behaviors observed were assessed without collaboration between observers.

Twenty-five behaviors (Table 1) were assessed following treatment or control procedures, in addition to monitoring the crest position (erect, lowered, or flat) and mentation (alert, quiet, depressed, or nonvigilant) of each cockatiel. Each observer evaluated 11 time points (0, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, and 9 hours postinjection or handling), for 10-minute periods, with every 10 minutes of video divided into 10 1-minute increments. The total number of observations for each of the 25 behaviors was tallied for each 10-minute observation period, with a notation on the crest position and mentation made at the end of each 10-minute period as well. Each evaluator observed each cockatiel for a total of 110 minutes.

Statistical analysis

Before the performance of statistical analysis, the total number of observations for each of the 25 behaviors recorded during the 10-minute observation periods for the 11 different time points was aggregated into a single total number per behavior for each observer over the total 110-minute observation window. Differences in observed behaviors between treatment and control were assessed using Wilcoxon sum rank test in a serial manner across each of the variables assessed (1 Wilcoxon test per variable) with a false discovery rate of 5%. A volcano plot plotting the fold changes between treatments and the P values from the Wilcoxon tests was performed to identify variables of interest. Fold changes were calculated based on medians. Correlation between variables was investigated using Spearman correlation coefficients in a correlation heatmap. Analysis was performed using statistical software (Metaboanalyst 5.0)27 for generating the correlation matrix. A P and q (latter = P value adjusted for false discovery rate) of .05 were used for statistical significance.

Agreement between the 3 observers was assessed using an intraclass correlation coefficient for a 2-way design for consistency among multiple raters through R (version 4.0.4; R Foundation for Statistical Computing).28 An intraclass correlation coefficient value of 0.81 to 1 was considered to have a strong level of agreement, a value of 0.61 to 0.80 to have a substantial level of agreement, a value of 0.41 to 0.60 to have a moderate level of agreement, a value of 0.21 to 0.40 to have a slight level of agreement, and a value lower than 0.20 to have a low level of agreement.

Results

For individual behaviors, focal preening (q = .023), burst preening (q = .036), and upright stance (q = .036) were significantly different between groups (Figure 1). Variables of interest (high magnitude of change coupled to statistical significance) were also visualized on the volcano plot. Variables of interest that displayed a large magnitude fold changes included: focal preening, burst preening, non–weight-bearing stance, holding body low and being nonvigilant (>2-fold increase in the carrageenan-injected birds), and upright stance (>2-fold increase in the control birds). There was an overall tendency for preening behaviors, both focal and burst, to be increased in the birds receiving carrageenan and for some of the more general behaviors (eating, drinking, stretching, etc) to not exhibit any specific trend. Correlation among scoring variables is plotted (Figure 2).

Figure 1
Figure 1

Volcano plot of the behavior variables with fold threshold of 2 (vertical dashed lines) and Wilcoxon sum-rank test threshold of P = .05 (horizontal dashed line) or q = .05 (horizontal dotted line). The further the data points are from the origin, the more important the variables are. Parameters on the top left corner are significant and 2-fold increased in treatment (carrageenan-injected birds), and the parameters on the top right corner are significant and 2-fold increased in control birds. Those variables falling on the left side of the “−1” vertical dashed line are increased with treatment (carrageenan-injected) birds, while those falling on the right side of the “1” vertical dashed line are increased with control birds. Types of scores are color coded. NWB = Non–weight-bearing.

Citation: American Journal of Veterinary Research 84, 10; 10.2460/ajvr.23.03.0052

Figure 2
Figure 2

Spearman correlation heatmap with hierarchical clustering illustrating variables that are moving in the same (red) or opposite (blue) directions together. Variables such as focal preening, NWB minute, and holding body low were more frequently demonstrated by treatment cockatiels, while variables such as biting cage, drinking, and time upright were appreciated more in control cockatiels. The color gauge on the right represents the Spearman correlation coefficient.

Citation: American Journal of Veterinary Research 84, 10; 10.2460/ajvr.23.03.0052

Agreement between the 3 observers was variable based on the behavior assessed, ranging from low to strong (Table 2). Behaviors with substantial to strong interobserver agreement included nonvigilant (“sleeping”) behavior, moving along cage floor, active climbing, moving along perch, thorough preening, rouse, scratch, stretch, eating, drinking, biting perch, biting cage bars, engaged with conspecific, upright stance, non–weight-bearing stance, and requiring bodily support. Behaviors found to have slight to low interobserver agreement included burst preening, focal preening, leaning, holding body low, lowered crest position, and flat crest position. All other behaviors not listed were found to have moderate interobserver agreement.

Table 2

Level of agreement between observers for behaviors assessed when evaluating treatment versus control cockatiels.

Variable ICC Interpretation
Bodily support 0.99 Strong
Upright 0.97 Strong
Drinking 0.95 Strong
Thorough preening 0.95 Strong
Active climbing 0.93 Strong
Eating 0.92 Strong
Lameness 0.92 Strong
NWB 0.89 Strong
Engage conspecific 0.87 Strong
Rouse 0.83 Strong
Stretch 0.83 Strong
Moving on perch 0.80 Substantial
Scratch 0.79 Substantial
Moving on floor 0.72 Substantial
Nonvigilant 0.70 Substantial
Biting perch 0.64 Substantial
Biting cage 0.61 Substantial
Perch resting 0.59 Moderate
Playing with toy 0.58 Moderate
Wipe beak 0.54 Moderate
Crest erect 0.54 Moderate
Interact with food 0.52 Moderate
Floor resting 0.47 Moderate
Focal preening 0.39 Slight
Burst preening 0.02 Low
Crest flat 0.0 Low
Leaning 0.0 Low
Holding body low −0.03 Low
Crest lowered −0.11 Low

An intraclass correlation coefficient (ICC) value of 0.81 to 1 was considered to have a strong level of agreement, value of 0.6 to 0.80 to have substantial level of agreement, value of 0.41 to 0.60 to have a moderate level of agreement, value of 0.21 to 0.40 to have a slight level of agreement, and value lower than 0.20 to have a low level of agreement. NWB = Non–weight-bearing.

Discussion

Evaluation of behavioral differences between control and treatment cockatiels in this study identified focal preening, burst preening, and upright stance to be significantly different between groups (Figure 1). When considering that the carrageenan model induces lameness through plantar foot inflammation and pain, effects such as decreased weight-bearing and increased preening of the injected limb were expected in treatment birds, whereas the upright stance was greater in the control birds likely due to them remaining sound on both limbs. It is interesting that significant increases in the number of instances of both burst preening and focal preening were appreciated in treatment birds, but instances of thorough preening did not significantly differ between treatment and control groups. Both burst preening and focal preening occur in relatively short duration, thereby allowing grooming behaviors without a prolonged decrease in overall vigilance of their environment. Similar observations of increased burst preening have been observed in cockatiels following exposure to stress (handling for physical examination and venipuncture),18 which supports the expectation that stress can often accompany pain, as experienced by the treatment cockatiels in this study. This difference is further highlighted in that interobserver agreement for burst preening and focal preening was low to slight, respectively, and significant differences in frequency were appreciated even with lower consistency. Additional behaviors that were reported to increase with stress in the study of Turpen et al18 included avoidance behaviors, crouching, resting, and plumage-shaking.

Behavioral correlations (Figure 2) identified behaviors that were more likely to be seen in control birds versus those more likely to be seen in treatment birds. Treatment cockatiels shared behaviors such as increased time spent non–weight-bearing and focal preening, more time holding their body low (as opposed to being upright), and more time spent nonvigilant. This contrasts with behaviors more likely to be expressed in control cockatiels such as time spent upright, drinking, and cage biting. In the context of their environment, cage biting and drinking from the water line can both be viewed as environmental interactions, with control cockatiels exhibiting more environmental interactions compared to the treatment group.

When identifying potential pain-related behaviors in a given species, it is important to understand how normal maintenance and social behaviors can be altered in the presence of stress without pain.14,15 Stress-related behavioral changes in cockatiels have been reported, which provided a comparison for the current study.18 Use of a control group in the current study additionally provided for observation of behavioral changes in the face of stress without concurrent pain present. When introduced to stress associated with handling and venipuncture in the study of Turpen et al,18 cockatiels had a significant decrease in “luxury” behaviors (locomotion, feeding, and environmental interactions) and a significant increase in resting and “reactionary” behaviors such as avoidance of objects/others and guarded crouching. In the current study, control cockatiels collectively demonstrated similar environmentally engaged activities such as cage biting and drinking from their water line, while treatment cockatiels were more likely to display behaviors such as being nonvigilant (sleeping). The cockatiel stress study18 reported that “maintenance” behaviors such as preening, stretching, and scratching did not significantly differ following the introduction of stress. This is in contrast to the current study, in which treatment cockatiels had a greater number of displays of focal and burst preening compared to control birds. The focal preening is likely associated with alterations to the limb caused by carrageenan injection in treatment cockatiels, given the minimal occurrence of focal preening in control birds. The significant increase in burst preening in carrageenan treatment birds is noteworthy because burst preening was noted in cockatiels posthandling with physical examination and venipuncture and therefore was assumed to be a stress-related behavior in the study of Turpen et al.18 Given the introduction of stress (handling) to both groups in our study, an initial assumption would be to have no significant difference in burst preening between control and treatment cockatiels. However, the significant difference in burst preening between the 2 groups highlights that burst preening may be an adaptive response of treatment cockatiels as a means of maintaining abbreviated grooming behaviors in the face of carrageenan-associated discomfort.

Other studies evaluating lameness (both induced and naturally occurring) in different avian species have reported significant behavioral differences between control and treatment birds. In chickens subjected to experimentally induced arthritis, common exhibitions of pain included behavioral changes such as 1-legged standing, overtly decreased mobility, increased sitting and laying down (ie, decreased activity), decreased grooming behaviors, reduction in the number of alert/observational head movements, reduction in eating and drinking behaviors, and decreased environmental pecking.2933 Various studies3443 evaluating musculoskeletal issues such as degenerative joint disorders and pododermatitis in turkeys and chickens found affected birds to more commonly display behaviors such as decreased walking speeds, shorter stride lengths, increased resting and tonic immobility, eating and drinking less, engaging less in maintenance behaviors such as preening, and even changes in social dynamics such as decreased group resting and slower initiations and engagements in sexual displays toward female conspecifics in turkeys. Also, Hispaniolan Amazon parrots and green-cheeked conures experimentally induced with arthritis displayed decreased weight-bearing on the affected limb, decreased ability to perch using both pelvic limbs, decreased ambulation and climbing abilities within their enclosures, changes in motivational behaviors to retrieve a hanging food reward, less time spent eating, and increased pecking at and overgrooming of their arthritic limb.4446

Behavioral observations were used to evaluate inflammatory pain in cockatiels in this study, with the intention that the information collected could be applied toward the future creation of an avian-specific pain scale useful for cockatiels and potentially other small psittacine species. However, one of the most challenging aspects of trying to create a pain scale is determining variables that can be readily recognized and assessed by a variety of observers in different settings. In the current study, greater than half of the behaviors had strong to substantial interobserver agreement between the 3 observers. Nevertheless, the level of agreement varied depending on the behavior observed. More dynamic behaviors such as active climbing, thorough preening, rousing, stretching, eating, and drinking had strong levels of interobserver agreement. Behaviors that required closer attention to detail or fell within similar categories, such as burst versus focal preening, leaning versus holding the body low, or having a flattened versus a lowered crest, had low levels of interobserver agreement. This may be indicative of difficulties in distinguishing these behaviors from one another, particularly if visibility of the bird is poor or if observers lack strong familiarity with the behaviors or species being observed. Dissonance in the knowledge base may have contributed to these findings as well, given that the ethogram was created by veterinarians but was applied by undergraduate student observers uniformly trained to recognize behaviors but lacking veterinary training. Despite this low level of interobserver agreement, several of these behaviors (burst and focal preening) were still found to be significant between control versus treatment birds, thereby further highlighting the importance of these behavioral differences. Although all observers were chosen based on past participation in animal behavioral studies, previous avian experience, and education in animal-related fields, the observations required to assess painful behaviors in this study may work most accurately and effectively with veterinary experience.

In this study, the type of pain evaluated focused on mild to moderate soft tissue inflammation and associated lameness. It is possible that some of the behavioral differences found are largely specific to focal, less severe pain in a foot (such as focal preening of the affected limb) but may be applicable to other types of lameness such as those associated with arthritis or orthopedic injuries. Classification of the severity of pain (mild, moderate, or severe) should be clearly defined using a given pain scale, as should the type of pain (acute vs chronic, nociceptive vs inflammatory vs neuropathic, etc). Assessment of an animal suspected to be in pain should encompass evaluating postural changes as well as any relevant facial changes, rather than focusing solely on the more expressive region of the face. This is even more important in birds since they have limited facial expression. As an example, the development of a Grimace scale for the rabbit47 has led to significant advancements in the recognition and treatment of pain in this species. However, in a separate study48 assessing various observers’ abilities to accurately identify rabbits in pain based on their facial features versus whole-body changes, observers were often unable to effectively identify painful individuals based on facial expression alone. Similarly, pain-related behavioral studies in cats have found that other areas of the body (tail, abdomen, limb positioning, etc) can be comparably expressive when evaluated concurrently with alterations in facial feature.10,49 As there are several parts of a bird in addition to the face that can become altered with behavioral changes, such as the tail, carriage of the wings, and head crests in those species that have them, these areas are worth further investigation. In this study, treatment cockatiels were more likely to hold their bodies low to the cage floor during observation periods, while control cockatiels held themselves more upright. Although crest position in this study did not significantly differ between groups, that may not hold true for other pain-related scenarios.

A focal continuous sampling method was used to evaluate behavioral differences between control and treatment groups. Continuous, “all-occurrences” sampling (as opposed to interval sampling) was chosen for this study given that an “all-occurrences” sampling method aims to provide an exact and faithful record of study behaviors via measurement of true, real-time frequencies.50 This allowed observers to evaluate a wide range of behaviors yet required approximately 110 minutes of video analysis for each of the 16 birds. Furthermore, because 25 unique behaviors plus crest position and mentation were analyzed for frequency despite some behaviors being longer in duration than others (thorough preening vs burst preening, etc), all behaviors were similarly weighted. Future studies removing behaviors found in this study to occur at very low frequencies or that did not significantly differ between groups would reduce the number of behaviors to observe, which may improve accuracy in measuring frequencies and allow duration measurements to be concurrently obtained. The use of behavior-measuring software such as BORIS (https://www.boris.unito.it/) may additionally improve the accuracy of assessments made but was unable to be applied in this study due to the large number of behaviors evaluated and the potential for confusion or frustration of the observers given the many key inputs that would have been needed.

The cockatiel was chosen as an ideal study subject to evaluate pain-related behaviors in this setting given this animal’s familiarity as a popular companion animal within psittacine species.16 If this type of behavioral study were to be further explored in future studies, administration of carrageenan in other psittacine species (both large and small) would be helpful in determining if similar behavioral responses are elicited postinjection. Differences in the anatomy of the foot, which could affect inflammatory responses can vary between psittacine species, let alone between avian orders, and may affect behavioral responses. For example, the foot is a crucial tool in podomandibulation and food consumption for larger psittacines, which differs from raptor species that use it for penetration of prey items and subsequent grasping of flesh. These types of birds could have additional noticeable effects as a result of carrageenan injection, such as decreased ability to grip perches or foot items and decreased appetite. Potential alterations in carrageenan-induced inflammatory patterns could also be affected by conformational differences in digital anatomy (zygodactyl vs anisodactyl vs tridactyl, etc) and the presence of additional distal limb features such as interdigital webbing (Anseriformes, Gruiformes, Sphenisciformes, etc).

Study limitations are acknowledged including that a single observer (NAM) performed all behavioral observations and created the ethogram used throughout the study. Having 1 initial observer may have introduced bias in terms of the behaviors recorded. However, the inclusion of 3 additional observers was utilized to minimize this bias and gather a more comprehensive assessment of behavioral differences. All 3 additional observers analyzed the video recordings with the same background knowledge regarding the study conditions, were trained in identical fashion by a single instructor (NAM), and viewed the videos in the same order and fashion to reduce bias in this subjective area of study. However, differences in overall knowledge base and species familiarity between observers may have contributed to underlying bias. Having the observers review the videos in the same order may have additionally reinforced false-positive outcomes. Randomization of video order was considered for further bias reduction but was ultimately elected against so as not to mask variables of interest given the smaller size of the study population. The single observer (NAM) was not included in the calculation of the interobserver agreement of this study. The relatively small number of birds per group and large number of individual behaviors assessed in this study may have contributed to a low power for each variable, as well as increased likelihood of false positive or negative associations between the treatment versus control group behaviors. Limitations in recording equipment capabilities lead to omission of behaviors such as vocalizations that could have varied between treatment and control cockatiels. Although beyond the scope of this article, evaluation of changes in individual vocalization associated with stress or discomfort may prove to be a useful observational tool when assessing pain in avian species. The cockatiels used in this study were able to visualize and hear one another throughout the study. Though this could have resulted in a heightened degree of stress during handling performed on each treatment and control bird, it was preferred over creating a permanent visual and auditory barrier with a psittacine species that is normally social and may have become stressed with prolonged isolation.

This study evaluated pain-related behaviors via use of a novel carrageenan-induced inflammatory model in cockatiels and found that focal preening, burst preening, and having an upright body stance each significantly differed between treatment versus control cockatiels. Additionally, this study had a strong level of interobserver agreement for the dynamic behaviors evaluated. Although there is still a paucity of information regarding accurate avian pain recognition and assessment, the findings obtained from this study can contribute to development of avian pain scales. Ongoing research regarding the behaviors of avian species is an essential field of interest and should be one that society continues to investigate to improve the well-being, medical care, and overall welfare of these animals.

Acknowledgments

We extend our gratitude to Shania Hamid, Elyza-Rose Ramirez, and Valeria Reyes Lua for their commitment to this study and the time spent assessing cockatiel behaviors with the research team. A special thank you is extended to Kristy Portillo and Daniel Pichardo for their direct support with husbandry and handling of the cockatiels throughout this project.

Disclosures

The authors do not have any conflicts of interest to disclose regarding this manuscript.

Funding

Funding for this research endeavor was provided by the Richard M. Schubot Parrot Wellness and Welfare Program, School of Veterinary Medicine, University of California-Davis.

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