investigated in chickens but the dose of butorphanol administered in the present study is similar to doses that have proved antinociceptive in other bird species [32–34]. It was not the aim of the present investigation to determine the optimal dose of butorphanol for analgesia in birds with keel fractures. It is possible that the mobility of hens with keel fractures would have been further increased by a different dose of butorphanol, depending on the relative balance between the potential for increased analgesic efficacy with a higher dose versus the potential for increased sedation. The pharmacokinetics of 2 mg/kg butorphanol IV have recently been investigated in broiler birds [41]. These data suggested that the onset of analgesia after butorphanol adminis- tration SC would be in the order of 30 minutes and that the duration of analgesia would be short, hence the timing of assessments after butorphanol administration in the present investigation. Morphine is analgesic in mammals and is widely used for provision of analgesia for acute pain states [42,43]. In chickens the antinociceptive effects of morphine, administered at varying doses, have been studied in acute models delivering noxious mechanical or thermal stimuli and both antinociception [44–46] and pronociception [47,48] has been reported. Morphine increased landing time in all the birds in the present study, an effect we attribute to sedation. Sleep-like behaviour and motor inco- ordination has been reported previously in birds following morphine [49,50]. The dose of morphine was chosen based on a recent pharmacokinetic study of morphine in broiler chickens [51] that reported only minor sedation following intravenous admin- istration of 2 mg/kg morphine. The profound sedation exhibited by our birds was, therefore, unexpected. In our experiment all birds were treated with morphine or saline first followed by butorphanol or saline eight days later. The rapid clearance of morphine in birds [51] suggests that there would have been no residual effects of morphine during the butorphanol phase of the study, confirmed by return of normal behaviour, without concurrent sedation, a few hours after morphine administration. Some healing of fractures is likely to have occurred over the eight day period, and it is possible that birds with fractures developed new additional fractures during the experimental period [12]. However fracture healing should have reduced the differential effects of butorphanol on the mobility of birds with and without keel fractures. Despite this, a significant interaction was still identified. No bird without fractures at the start of the study developed a fracture during the course of the experiment, but it was impossible to determine at post-mortem whether birds with fractures at the outset developed new fractures. By the butorpha- nol phase of the study, birds were more familiar with the test procedure, evidenced by their shorter latencies to land from all three perch heights after saline injection in the butorphanol phase than after saline treatment in the morphine phase. Although the data for morphine and butorphanol were analysed independently of each other we cannot discount the possibility that prior exposure to morphine confounded the effects of butorphanol administration to birds with keel fractures, influencing our experimental results. The investigator was unaware of the fracture status of individual birds at the time of carrying out the landing test, but was not blinded to the treatment received by each bird (opioid or saline). Blinding to drug treatment would have been difficult because of the obvious behavioural effects caused by morphine administration to the hens. However because latency to land is an objective measure of bird mobility it is unlikely that the absence of blinding biased the data generated in this experiment. Laying Hens and Keel Fractures Conclusions This is the first study to provide a solid evidential base that birds with keel fractures experience pain, a finding that has significant implications for the welfare of laying hens that are housed in non- cage or furnished caged systems. It is not appropriate or achievable to provide analgesia to birds with keel fractures in commercial housing systems. Rather the results of this study provide a compelling argument to prevent the development of keel fractures via genetic selection for more resilient bones, modified feeding and production practices and/or improved housing system design. Methods Ethics statement The study was carried out under the Animals (Scientific Procedures) Act (PPL 30/2865). For experiments carried out under Home Office License local ethical approval by the institution is not required. Birds and management Sixty two Lohman Brown strain of laying hens (aged 35 weeks) were obtained from a commercial farm; they were all assessed by palpation of the keel [1] to detect those with suspected healed keel fractures. Twenty-eight hens were selected with keel fractures, and 34 without evidence of fractures were selected as controls. They were randomly divided into two groups, each containing a similar number of hens with or without fractures. Hens were weighed on arrival at the experimental site. After allowing three days acclimatisation at the experimental site, the experiment was started for group 1 (31 hens), while group 2 did not undergo testing until 10 days after arrival, the experimental protocol was identical for both groups of hens. Each group was housed in a floor pen (3 m63.5 m). Study protocol We subjected the birds to a landing test [13] to investigate whether the test drugs altered the time the hens took to fly down from different perch heights to the ground to access a food reward. All the hens were initially trained by placing them on a low perch (20 cm) and allowing them to see a meal-worm. Hens were considered trained when they jumped from this low perch in less than 10 s and ate the food reward. Four birds (2 from each group) could not be trained to complete the landing test. Trained hens in each group were randomly allocated to receive treatment with either morphine or saline first. Hens that received the morphine treatment were injected subcutaneously in the dorsal neck with morphine 2 mg/kg (morphine sulphate, 10 mg/ml solution) while the remaining hens were injected with an equivalent volume of saline. The hens were left for 30 minutes before starting the landing test. Each landing test comprised three trials with the perch set at 50 cm, 100 cm and finally 150 cm from the ground. After a 1 day rest period the landing test repeated following a second treatment with either morphine or saline. Birds that received morphine as the first treatment received saline as the second treatment, while birds that received saline first were treated with morphine. This sequence was then repeated such that each hen completed the landing test twice with morphine and twice with saline in a cross-over design. A rest interval of 8 days after final morphine administration was allowed to ensure that the drug was completely metabolised and cleared from their system. Following the rest interval, the test procedure was repeated to examine the effect of butorphanol (2 mg/kg (Torbugesic 1% W/V PLOS ONE | www.plosone.org 4 August 2012 | Volume 7 | Issue 8 | e42420