thermal-imaging-to-study-stress-non-invasively-in-unrestrained-birds

Thermal Imaging to Study Stress Non-invasively in Unrestrained Birds

Stress, a central concept in biology, describes a suite of emergency responses to challenges. Among other responses, stress leads to a change in blood ...

University of Glasgow

Extracellular single unit recordings were made from various depths in the left olfactory bulb of ten anaesthetised, freely breathing adult hens (Gallus domesticus) using glass insulated tungsten microelectrodes. The 66 spontaneously active neurons recorded had widely variable firing rates (mean 4.9 spikes/s, range 0.1-32.4 spikes/s) and variable temporal firing patterns (regular, bursting and random discharge). Interspike interval histograms were constructed for each unit and tested for goodness of fit to theoretical distributions. The activity of 23 units fitted gamma distributions, six units fitted lognormal distributions, three units fitted Weibull distributions, one unit fitted an exponential distribution and 11 units had bimodal distributions. Responses of some units to odour stimuli (Clove oil, Geraniol, Limonene and Ammonia all at 10% vapour saturation) delivered directly to the olfactory epithelium were examined. Odour stimulation modified spontaneous activity in 29 of the 44 units tested, causing either inhibition (56%) or excitation (44%) of firing. Of the 35 units stimulated with more than one odour, 15 responded to multiple stimuli, showing excitation or inhibition only or both inhibition and excitation, depending on the odour applied. Ammonia most commonly elicited a response (70% of applications) and all the odours were capable of inducing both inhibition and excitation of spontaneous firing. Examination of transverse sections of adult hen olfactory bulb in the region corresponding to the recording sites allowed unit recording depth to be related to cellular layers. There was some evidence of different neurone properties in each bulb layer. The spontaneous activity and odour responses observed resemble those seen in other vertebrate groups and the firing rates observed are between those previously reported for mammals and reptiles. This study provides detailed physiological evidence for a well-developed functional olfactory system in an avian species.

Spontaneous and odour evoked activity in single avian olfactory bulb neurones.

Although the chemoreceptive properties of the nasal trigeminal system are well known, the physiological characteristics of nasal nociceptors responding to both mechanical and chemical stimulation have not been well described. In this study, the activity of single nasal trigeminal units recorded from microdissected twigs of the ethmoidal nerve of anaesthetized hens (Gallus domesticus) was investigated. Using a mechanical search stimulus, 20 slowly and 22 rapidly adapting nasal mechanoreceptors were identified, exhibiting mean thresholds of 2.96 g. Twelve slowly adapting units also exhibited chemical sensitivity when exposed to ammonia gas. These had mean response thresholds of 0.232% vapour saturation and exhibited variable stimulus-response profiles. This is the first study to quantify the responses of polymodal nasal nociceptors to a noxious airborne chemical in any species.

Response characteristics of nasal trigeminal nociceptors in Gallus domesticus.

This study investigated olfactory processing in a functional context by examining the responses of single avian olfactory bulb neurones to two biologically important gases over relevant concentration ranges. Recordings of extracellular spike activity were made from 80 single units in the left olfactory bulb of 11 anaesthetised, freely breathing adult hens (Gallus domesticus). The units were spontaneously active, exhibiting widely variable firing rates (0.07-47.28 spikes/s) and variable temporal firing patterns. Single units were tested for their response to an ascending concentration series of either ammonia (2.5-100 ppm) or hydrogen sulphide (1-50 ppm), delivered directly to the olfactory epithelium. Stimulation with a calibrated gas delivery system resulted in modification of spontaneous activity causing either inhibition (47% of units) or excitation (53%) of firing. For ammonia, 20 of the 35 units tested exhibited a response, while for hydrogen sulphide, 25 of the 45 units tested were responsive. Approximate response thresholds for ammonia (median threshold 3.75 ppm (range 2.5-60 ppm, n=20)) and hydrogen sulphide (median threshold 1 ppm (range 1-10 ppm, n=25)) were determined with most units exhibiting thresholds near the lower end of these ranges. Stimulus response curves were constructed for 23 units; 16 (the most complete) were subjected to a linear regression analysis to determine whether they were best fitted by a linear, log or power function. No single function provided the best fit for all the curves (seven were linear, eight were log, one was power). These findings show that avian units respond to changes in stimulus concentration in a manner generally consistent with reported responses in mammalian olfactory bulb neurones. However, this study illustrates a level of fine-tuning to small step changes in concentration (<5 ppm) not previously demonstrated in vertebrate single olfactory bulb neurones.

Stimulus-response functions of single avian olfactory bulb neurones.

Adaptation in the avian olfactory bulb (OB) was examined by recording the activity of single OB neurones in the chicken (Gallus domesticus) during prolonged (120 s) odour exposure (20 ppm ammonia). The activity of each neurone before, during and after odour stimulation was investigated with analysis examining changes in firing rate and pattern. Of the 73 OB neurones recorded, 22 responded to ammonia stimulation with either inhibition or excitation of firing (both 50%). Their responses then either adapted (lessening of the response, 32%) or sensitized (heightening of the response, 68%) during the odour stimulus period. Both inhibited and excited units underwent adaptation or sensitization and their responses were consistent during odour stimulation. These results demonstrate the role of OB neurones in adaptive responses of the avian olfactory system during prolonged odour stimulation.

Adaptation responses of single avian olfactory bulb neurones.

This study characterised and compared the properties of chemically sensitive nociceptors in the avian nasal and oral epithelia recorded from either microelectrode stabs of the trigeminal ganglion (nasal mucosa) or microdissected twigs of the nasopalatine nerve (palatine mucosa) in urethane anaesthetized hens. A total of 146 slowly and rapidly adapting nasal and palate mechanically sensitive receptors were identified exhibiting von Frey thresholds to mechanical stimulation ranging from 0.2 to 8.3 g (mean 3.21+/-2.28 g, n=39) in the nasal cavity and <0.1 to 15 g (mean 2.17+/-2.69 g, n=62) in the palate. Receptive fields were situated throughout the nasal cavity and palate. A subset of receptors (classified as polymodal nociceptors) exhibited chemical sensitivity when exposed to noxious levels of ammonia gas, and some polymodal afferents also responded to acetic acid vapour or carbon dioxide exposure. Responses to ammonia were characterised by delayed onset, rapid prolonged discharge patterns usually outlasting the chemical stimulation. Clear concentration-response relationships were apparent for chemically receptive afferents in both the nasal and oral cavities, although curve shape varied considerably between receptors. Statistical comparisons of the responses of polymodal nociceptors in each mucosal region revealed no significant difference between their mechanical thresholds, but nasal afferents exhibited lower chemical (ammonia) thresholds and higher maximal firing rates. These response characteristics highlight distinct sensitivities to potentially painful chemical stimulation between epithelial areas. These effects may be due to the inherent properties of the nociceptors in each region but are also likely to reflect the role of the perireceptor environment in mediating peripheral nociception.

Mechano-chemical nociceptors in the avian trigeminal mucosa.

Despite growing evidence of avian olfactory abilities, there are few reports of behavioral correlates of chemosensory stimulation in birds. The present study aimed to determine how the behavioral responses of hens to selected gases might be affected by input from different chemosensory systems. We also hoped to relate electrophysiological thresholds previously measured in our laboratory to behavioral evidence of perception. Immediate behavioral responses to ascending series of short (7 s) pulses of olfactory (hydrogen sulphide), trigeminal (carbon dioxide) and combining (ammonia) gaseous stimulants were measured in 12 partially restrained adult hens (Gallus domesticus) using a purpose built gas delivery system. The concentration ranges applied matched those used in our previous electrophysiological studies of olfactory bulb responses. Consistent and specific behavioral responses were observed in response to stimulation with each gas. While significant increases in mandibulation (a distinctive rapid bout of bill movements), interruption of ongoing behavior, orientation to the end of the stimulus, and struggling were seen during stimulation with all three gases, orientation towards the stimulus occurred only in response to the olfactory stimulants, ammonia and hydrogen sulphide. The only gas to elicit significant avoidance was hydrogen sulphide, while gasping and headshaking were elevated in response to stimulation with carbon dioxide. Approximate threshold values for some types of behavior related reasonably well to receptor thresholds determined electrophysiologically, but perception may have occurred at lower concentrations without overt behavioral consequences. The diversity of the behavioral responses observed supports the notion that the gases selectively stimulated different sensory pathways.

Behavioral correlates of olfactory and trigeminal gaseous stimulation in chickens, Gallus domesticus.

The first detailed physiological evidence for olfactory and trigeminal chemoreception in an avian species is provided by a series of investigations in the chicken (Gallus domesticus). Initial work indicated that the activity of avian olfactory bulb neurones closely resembles that of other vertebrates, exhibiting variable spontaneous temporal firing patterns with mean firing rates between those reported for mammals and reptiles. Application of odors directly to the olfactory epithelium showed that like mammals, avian olfactory bulb neurones respond in the form of inhibition and excitation with accompanying changes in temporal firing pattern. When exposed to a range of concentrations of a single odor, all responsive neurones exhibited an ability to discriminate small step-changes in concentration producing clear stimulus response relationships. Avian trigeminal chemoreception was also investigated by examining the responses of single mucosal receptors in the nasal cavity and palate. Slowly and rapidly adapting nasal mechanoreceptors were identified, some of which exhibited chemical sensitivity when exposed to ammonia gas, acetic acid vapor or carbon dioxide. These results demonstrate that polymodal nociceptors are present in avian nasal mucosa and represent the first attempt in any species to quantify the responses of single trigeminal receptors to a range of concentrations of noxious airborne chemicals. Collectively, the findings demonstrate how an electrophysiological approach can improve our understanding of the underlying sensory physiology relating to avian perception of the chemical environment.

Avian chemoreception: an electrophysiological approach.

Pain associated with poultry lameness is poorly understood. The anti-nociceptive properties of two non-steroidal anti-inflammatory drugs (NSAIDs) were evaluated using threshold testing in combination with an acute inflammatory arthropathy model. Broilers were tested in six groups (n=8 per group). Each group underwent a treatment (saline, meloxicam (3 or 5mg/kg) or carprofen (15 or 25mg/kg)) and a procedure (Induced (arthropathy-induction) or sham (sham-handling)) prior to testing. Induced groups had Freund's complete adjuvant injected intra-articularly into the left intertarsal joint (hock). A ramped thermal stimulus (1°C/s) was applied to the skin of the left metatarsal. Data were analysed using random-intercept multi-level models. Saline-induced birds had a significantly higher skin temperature (± SD) than saline-sham birds (37.6 ± 0.8°C vs. 36.5 ± 0.5°C; Z=-3.47, P<0.001), consistent with an inflammatory response. Saline was associated with significantly lower thermal thresholds (TT) than analgesic treatment (meloxicam: Z=2.72, P=0.007; carprofen: Z=2.58, P=0.010) in induced birds. Saline-induced birds also had significantly lower TT than saline-sham birds (Z=-2.17, P=0.030). This study found direct evidence of an association between inflammatory arthropathies and thermal hyperalgesia, and showed that NSAID treatment maintained baseline thermal sensitivity (via anti-nociception). Quantification of nociceptive responsiveness in a predictable broiler pain model identified thermal anti-hyperalgesic properties of two NSAIDs, which suggested that therapeutically effective treatment was provided at the doses administered. Such validation of analgesic strategies will increase the understanding of pain associated with specific natural broiler lameness types.

Thermal nociception as a measure of non-steroidal anti-inflammatory drug effectiveness in broiler chickens with articular pain.

Defining states of clinical consciousness in animals is important in veterinary anaesthesia and in studies of euthanasia and welfare assessment at slaughter. The aim of this study was to validate readily observable reflex responses in relation to different conscious states, as confirmed by EEG analysis, in two species of birds under laboratory conditions (35-week-old layer hens (n=12) and 11-week-old turkeys (n=10)). We evaluated clinical reflexes and characterised electroencephalograph (EEG) activity (as a measure of brain function) using spectral analyses in four different clinical states of consciousness: conscious (fully awake), semi-conscious (sedated), unconscious-optimal (general anaesthesia), unconscious-sub optimal (deep hypnotic state), as well as assessment immediately following euthanasia. Jaw or neck muscle tone was the most reliable reflex measure distinguishing between conscious and unconscious states. Pupillary reflex was consistently observed until respiratory arrest. Nictitating membrane reflex persisted for a short time (<1 min) after respiratory arrest and brain death (isoelectric EEG). The results confirm that the nictitating membrane reflex is a conservative measure of death in poultry. Using spectral analyses of the EEG waveforms it was possible to readily distinguish between the different states of clinical consciousness. In all cases, when birds progressed from a conscious to unconscious state; total spectral power (PTOT) significantly increased, whereas median (F50) and spectral edge (F95) frequencies significantly decreased. This study demonstrates that EEG analysis can differentiate between clinical states (and loss of brain function at death) in birds and provides a unique integration of reflex responses and EEG activity.

Avian reflex and electroencephalogram responses in different states of consciousness.

Acute stress triggers peripheral vasoconstriction, causing a rapid, short-term drop in skin temperature in homeotherms. We tested, for the first time, whether this response has the potential to quantify stress, by exhibiting proportionality with stressor intensity. We used established behavioural and hormonal markers: activity level and corticosterone level, to validate a mild and more severe form of an acute restraint stressor in hens (Gallus gallus domesticus). We then used infrared thermography (IRT) to non-invasively collect continuous temperature measurements following exposure to these two intensities of acute handling stress. In the comb and wattle, two skin regions with a known thermoregulatory role, stressor intensity predicted the extent of initial skin cooling, and also the occurrence of a more delayed skin warming, providing two opportunities to quantify stress. With the present, cost-effective availability of IRT technology, this non-invasive and continuous method of stress assessment in unrestrained animals has the potential to become common practice in pure and applied research.

Dorothy McKeegan

Institute of Biodiversity,
Animal Health and Comparative Medicine,
College of Medical,
Veterinary & Life Sciences,
Institute of Biodiversity, Animal Health and Comparative Medicine, College of Medical, Veterinary & Life Sciences

University of Glasgow

Thermal Imaging to Study Stress Non-invasively in Unrestrained Birds