solution for injection) injected subcutaneously in the dorsal neck) or saline administration in a second cross over design experiment. As before, the injected hens were left for 30 minutes before starting the test. Each hen completed the landing test twice with butorphanol and twice with saline, with a 1 day rest period allowed to elapse between administration of either butorphanol or saline. The mean time taken to land was calculated for each drug and for saline for each bird, prior to statistical analysis. Thirty eight days (group 1) and 48 days (group 2) after arrival birds were killed humanely using a Schedule 1 method and each was examined post-mortem to determine the extent and nature of any keel bone pathology. If keel fractures were detected their severity was rated as either mild (severity score 1) or severe (severity score 2) [12]. References 1. Wilkins LJ, Brown SN, Zimmerman PH, Leeb C, Nicol CJ (2004) Investigation of palpation as a method for determining the prevalence of keel and furculum damage in laying hens. Vet Rec 155: 547–549. 2. Wilkins LJ, McKinstry JL, Avery NC, Knowles TG, Brown SN, et al. (2011) Influence of housing system and design on bone strength and keel bone fractures in laying hens. Vet Rec 169: 414. 3. Bove SE, Flatters SJ, Inglis JJ, Mantyh PW (2009) New advances in musculoskeletal pain. Brain Res Rev 60: 187–201. 4. Freeman KT, Koewler NJ, Jimenez-Andrade JM, Buus RJ, Herrera MB, et al. (2008) A fracture pain model in the rat: adaptation of a closed femur fracture model to study skeletal pain. Anesthesiology 108: 473–483. 5. de Mos M, de Bruijn AG, Huygen FJ, Dieleman JP, Stricker BH, et al. (2007) The incidence of complex regional pain syndrome: a population-based study. Pain 129: 12–20. 6. Santy J, Mackintosh C (2001) A phenomenological study of pain following fractured shaft of femur. J Clin Nurs 10: 521–527. 7. Woolf AD, Pfleger B (2003) Burden of major musculoskeletal conditions. Bull World Health Organ 81: 646–656. 8. Jimenez-Andrade JM, Bloom AP, Mantyh WG, Koewler NJ, Freeman KT, et al. (2009) Capsaicin-sensitive sensory nerve fibers contribute to the generation and maintenance of skeletal fracture pain. Neuroscience 162: 1244–1254. 9. Capner CA, Lascelles BD, Waterman-Pearson AE (1999) Current British veterinary attitudes to perioperative analgesia for dogs. Vet Rec 145: 95–99. 10. Hugonnard M, Leblond A, Keroack S, Cadore JL, Troncy E (2004) Attitudes and concerns of French veterinarians towards pain and analgesia in dogs and cats. Vet Anaesth Analg 31: 154–163. 11. Waran N, Williams VM, Clarke N, Bridge IS (2010) Recognition of pain and use of analgesia in horses by veterinarians in New Zealand. N Z Vet J 58: 274–280. 12. Richards GJ, Nasr MA, Brown SN, Szamocki EM, Murrell J, et al. (2011) Use of radiography to identify keel bone fractures in laying hens and assess healing in live birds. Vet Rec 169: 279. 13. Nasr MAF, Murrell J, Wilkins LJ, Nicol CJ (2012) The effect of keel fractures on egg-production parameters, mobility and behaviour in laying hens. Anim Welfare 21: 127–135. 14. Brown DC, Boston RC, Farrar JT (2010) Use of an activity monitor to detect response to treatment in dogs with osteoarthritis. J Am Vet Med Assoc 237: 66– 70. 15. Weaver LA, Blaze CA, Linder DE, Andrutis KA, Karas AZ (2010) A model for clinical evaluation of perioperative analgesia in rabbits (Oryctolagus cuniculus). J Am Assoc Lab Anim Sci 49: 845–851. 16. Nagase H, Kumakura S, Shimada K (2012) Establishment of a novel objective and quantitative method to assess pain-related behavior in monosodium iodoacetate-induced osteoarthritis in rat knee. J Pharmacol Toxicol Methods 65: 29–36. 17. Flecknell PA (2010) Do mice have a pain face? Nat Methods 7: 437–438. 18. May A (2007) Neuroimaging: visualising the brain in pain. Neurol Sci 28 Suppl 2: S101–107. 19. Liang M, Mouraux A, Iannetti GD (2011) Parallel processing of nociceptive and non-nociceptive somatosensory information in the human primary and secondary somatosensory cortices: evidence from dynamic causal modeling of functional magnetic resonance imaging data. J Neurosci 31: 8976–8985. 20. Silverman J, Garnett NL, Giszter SF, Heckman CJ, 2nd, Kulpa-Eddy JA, et al. (2005) Decerebrate mammalian preparations: unalleviated or fully alleviated pain? A review and opinion. Contemp Top Lab Anim Sci 44: 34–36. 21. Sufka KJ, Weed NC (1994) Construct validation of behavioral indices of isolation stress and inflammatory nociception in young domestic fowl. Physiol Behav 55: 741–746. 22. Gentle MJ, Bradbury JM, Wilson S (2003) Sensory properties of articular afferents following Mycoplasma arthritis in the chicken. Brain Res 968: 26–34. Laying Hens and Keel Fractures Statistical analysis Statistical analysis on the mean time taken to land was carried out using PASW Statistics (SPSS version 18.0 for Windows). For each perch height, a General Linear Model Repeated Measures design was used. We examined the effect of two between-subjects factors (i) the keel fracture status of the bird (fracture/no fracture) and (ii) the order of drug presentation (butorphanol tested before or after saline). The effect of butorphanol or saline administration was examined as the repeated measure for each bird. Values were considered significant at p#0.05. Author Contributions Conceived and designed the experiments: MAFN CJN JM. Performed the experiments: MAFN. Analyzed the data: MAFN CJN. Contributed reagents/materials/analysis tools: CJN JM. Wrote the paper: MAFN CJN JM. 23. McKeegan DE (2004) Mechano-chemical nociceptors in the avian trigeminal mucosa. Brain Res Brain Res Rev 46: 146–154. 24. Gentle MJ (1997) Pain-related behaviour following sodium urate arthritis is expressed in decerebrate chickens. Physiol Behav 62: 581–584. 25. Gentle MJ, Hunter LN, Corr SA (1997) Effects of caudolateral neostriatal ablations on pain-related behaviour in the chicken. Physiol Behav 61: 493–498. 26. Hawkins MG, Paul-Murphy J (2009) Avian analgesia. Vet Clin North Am Exot Anim Pract 14: 61–80. 27. Jarvis ED, Gunturkun O, Bruce L, Csillag A, Karten H, et al. (2005) Avian brains and a new understanding of vertebrate brain evolution. Nat Rev Neurosci 6: 151–159. 28. Hocking PM, Gentle MJ, Bernard R, Dunn LN (1997) Evaluation of a protocol for determining the effectiveness of pretreatment with local analgesics for reducing experimentally induced articular pain in domestic fowl. Res Vet Sci 63: 263–267. 29. Gentle MJ, Hocking PM, Bernard R, Dunn LN (1999) Evaluation of intraarticular opioid analgesia for the relief of articular pain in the domestic fowl. Pharmacol Biochem Behav 63: 339–343. 30. Hocking PM, Robertson GW, Gentle MJ (2001) Effects of anti-inflammatory steroid drugs on pain coping behaviours in a model of articular pain in the domestic fowl. Res Vet Sci 71: 161–166. 31. Hocking PM, Robertson GW, Gentle MJ (2005) Effects of non-steroidal anti- inflammatory drugs on pain-related behaviour in a model of articular pain in the domestic fowl. Res Vet Sci 78: 69–75. 32. Paul-Murphy JR, Brunson DB, Miletic V (1999) Analgesic effects of butorphanol and buprenorphine in conscious African grey parrots (Psittacus erithacus erithacus and Psittacus erithacus timneh). Am J Vet Res 60: 1218–1221. 33. Sladky KK, Krugner-Higby L, Meek-Walker E, Heath TD, Paul-Murphy J (2006) Serum concentrations and analgesic effects of liposome-encapsulated and standard butorphanol tartrate in parrots. Am J Vet Res 67: 775–781. 34. Paul-Murphy JR, Krugner-Higby LA, Tourdot RL, Sladky KK, Klauer JM, et al. (2009) Evaluation of liposome-encapsulated butorphanol tartrate for alleviation of experimentally induced arthritic pain in green-cheeked conures (Pyrrhura molinae). Am J Vet Res 70: 1211–1219. 35. Witter J, Dionne RA (2004) What can chronic arthritis pain teach about developing new analgesic drugs? Arthritis Res Ther 6: 279–281. 36. McGeown D, Danbury TC, Waterman-Pearson AE, Kestin SC (1999) Effect of carprofen on lameness in broiler chickens. Vet Rec 144: 668–671. 37. Hothersall B, Caplen G, Nicol CJ, Waterman-Pearson AE, Weeks CA, et al. Challenges of determining links between pain and lameness in broiler chickens; 2012; York, UK. 38. Commiskey S, Fan LW, Ho IK, Rockhold RW (2005) Butorphanol: effects of a prototypical agonist-antagonist analgesic on kappa-opioid receptors. J Pharmacol Sci 98: 109–116. 39. Mansour A, Khachaturian H, Lewis ME, Akil H, Watson SJ (1988) Anatomy of CNS opioid receptors. Trends Neurosci 11: 308–314. 40. Guzman DS, Flammer K, Paul-Murphy JR, Barker SA, Tully TN, Jr. (2011) Pharmacokinetics of butorphanol after intravenous, intramuscular, and oral administration in Hispaniolan Amazon parrots (Amazona ventralis). J Avian Med Surg 25: 185–191. 41. Singh PM, Johnson C, Gartrell B, Mitchinson S, Chambers P (2011) Pharmacokinetics of butorphanol in broiler chickens. Vet Rec 168: 588. 42. Robertson SA, Taylor PM (2004) Pain management in cats–past, present and future. Part 2. Treatment of pain–clinical pharmacology. J Feline Med Surg 6: 321–333. 43. Costantini R, Affaitati G, Fabrizio A, Giamberardino MA (2011) Controlling pain in the post-operative setting. Int J Clin Pharmacol Ther 49: 116–127. 44. Schneider C (1961) Effects of morphine-like drugs in chicks. Nature 191: 607– 608. PLOS ONE | www.plosone.org 5 August 2012 | Volume 7 | Issue 8 | e42420