that walking and flying become mechanically impaired by an anatomical deformity of the keel bone and damage to the pectoralis major and supracoriacoideus muscles that insert on it, causing subsequent muscle degeneration or disuse atrophy. The keels of birds with healed keel fractures were colder than those of birds with normal keels, assessed using infra-red thermography [13], supporting this hypothesis. However it is also possible that if birds have a capacity to feel pain, then ongoing pain resulting from fracture of the keel bone could contribute to decreased mobility. Pain may be inferred in animals by observing changes in their behaviour, either the cessation of abnormal behaviours or the resumption of normal behaviours when drugs that are known to be analgesic in man are administered [14–16]. The aim of this study was to provide proof of concept that the administration of appropriate analgesic drugs might reverse the impairment of mobility observed in birds with keel fracture. Such a result would contribute to the general debate about the capacity of birds to experience pain, as well as to the specific debate about whether fractures are associated with chronic pain in laying hens. Two different classes of opioids, a kappa agonist butorphanol and the full mu agonist morphine, were administered to two groups of birds, one with and one without keel fractures and their mobility was assessed. Results The mean (SEM) bodyweights of birds with and without keel bone fractures on arrival at the experimental site were 1.79 (0.02) and 1.82 (0.03) kg respectively, this difference was not statistically significant (p.0.05). For all hens, keel bone fracture status was reclassified post mortem following keel bone dissection. We found 23 birds without keel bone fracture and 35 birds with a keel bone fracture. Morphine Morphine increased the time to land in all the birds, whether they had fractures or not, from the 100 and 150 cm perch heights, but not from 50 cm, compared to saline. Overall, birds treated with morphine took approximately 3 times longer to fly down from 100 cm perches (mean (SEM) landing time (sec) 32.0 (10.1) morphine, 11.6 (5.2) saline, F = 10.08, p,0.001) and 1.5 times longer to fly down from 150 cm perches (mean (SEM) landing time (sec) 49.5 (11.8) morphine, 34.0 (9.8) saline, F = 3.89, p = 0.05). In addition, birds with keel fractures took approximately 6 times longer to fly down from the 100 cm perches (mean (SEM) landing time (sec) 6.6 (11.4) birds without keel fractures, 37.0 (9.3) birds with keel fractures, F = 4.25, p = 0.04) and 8 times longer to fly down from the 150 cm perches, (mean (SEM) landing time (sec) 9.9 (15.7) birds without keel fractures, 73.6 (12.8) birds with keel fractures, F = 9.88, p,0.001) than hens without fractures, although there was no statistical difference with the low, 50 cm perch. No interaction between the effects of morphine on landing time and the presence of a fracture was found for any perch height. There was no significant effect of order of drug treatment (whether morphine or saline was administered first) on landing time (p.0.05). Morphine caused the birds to become sedated, and when not stimulated, the birds adopted sleep like postures. Butorphanol There was a significant interaction between presence or absence of a keel fracture and the effects of butorphanol at all perch heights (50 cm: F = 3.71 p = 0.05; 100 cm: F = 4.72, p = 0.03; 150 cm F = 5.53, p = 0.02; df 1,54 in all cases). In hens with keel fractures, butorphanol decreased the latency to land from the different perch Laying Hens and Keel Fractures heights by approximately 20% compared with the latencies recorded for these hens following saline (Figure 1). However even with butorphanol treatment the latency to land remained significantly greater for hens with fractures than hens without. Butorphanol administration to hens without keel fractures either marginally increased (50 cm and 150 cm perch heights) or decreased (100 cm perch height) latency to land, but the magnitude of these effects were minimal and unlikely to be of any biological significance compared to the decrease in latency shown by hens with keel fractures. There was no significant effect of order of drug treatment (saline or butorphanol first) on landing time (p.0.05). Butorphanol did not cause any obvious effects on spontaneous behaviour and no sedation was apparent. Post mortem of birds at the end of the study confirmed that all birds were correctly attributed to group (fracture or no fracture), and no birds classified without fractures at the start of the study developed a fracture during the course of the experiment. The average keel bone fracture severity score was 1.94. Discussion Due to the unique qualities of pain as both a sensory and emotional experience, the ability of animals to experience pain, as opposed to nociception, cannot be ascertained without doubt. However there is some acceptance within the scientific community that non-human mammals might be capable of experiencing pain [17]. This is attributed in part to the similarities in the central nervous system between human and non-human mammals, although these similarities in brain structure alone do not provide sufficient evidence for the capacity of non-human mammals to experience pain. In humans, functional imaging and electroen- cephalographic studies consistently show activation of defined cerebral cortical structures during the administration of stimuli reported to be painful by the experimental subject, for example the primary and secondary somatosensory cortices, amygdala, ante- rior cingulated cortex and insula [18]. However these structures are also active during processing of non-painful, but salient, stimuli, such as auditory or visual cues [19], therefore the specific neural mechanisms leading to the experience of pain remain to be elucidated. Despite this uncertainty, it is widely acknowledged that an intact cerebral cortex is required for pain perception in mammals [20]. In contrast, although birds possess the neuroanatomical pathways necessary for nociception [21–23] there are opposing views as to the capacity of birds to experience pain [24–26]. An intact pallium, the functional equivalent of the cerebral cortex of mammals [27] may be a necessary prerequisite for birds to have the capacity to experience pain. However, the existence of an intact pallium alone does not provide sufficient evidence of pain capacity. Further evidence can be obtained by examining the effect of administration of drugs with known antinociceptive properties. When arthritis is experimentally induced (by injection of sodium urate into the legs of chickens), behaviours such as limb guarding are increased, whilst normal locomotry and feeding behaviours are reduced [28–31]. Administration of intra-articular bupivicaine [28] systemic steroids [30] or non steroidal anti-inflammatory drugs (NSAIDs) [31] restored the frequency of some of these behaviours to control values. In other bird species, butorphanol has also proved antinociceptive in experimentally induced arthritis [32–34]. Collectively these results suggest that birds have the capacity to experience pain, although caution is required. Gentle et al. (1997) [24] found that decerebrate birds showed the same repertoire of ‘‘pain’’ related behaviours as intact birds. This PLOS ONE | www.plosone.org 2 August 2012 | Volume 7 | Issue 8 | e42420