UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Behavioural and physiological responses of Steller sea lions to invasive marking techniques : evidence… Walker, Kristen Amy 2010

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
24-ubc_2010_fall_walker_kristen.pdf [ 1.74MB ]
Metadata
JSON: 24-1.0071136.json
JSON-LD: 24-1.0071136-ld.json
RDF/XML (Pretty): 24-1.0071136-rdf.xml
RDF/JSON: 24-1.0071136-rdf.json
Turtle: 24-1.0071136-turtle.txt
N-Triples: 24-1.0071136-rdf-ntriples.txt
Original Record: 24-1.0071136-source.json
Full Text
24-1.0071136-fulltext.txt
Citation
24-1.0071136.ris

Full Text

BEHAVIOURAL AND PHYSIOLOGICAL RESPONSES OF STELLER SEA LIONS TO INVASIVE MARKING TECHNIQUES: EVIDENCE OF POST-OPERATIVE PAIN by KRISTEN AMY WALKER B.Sc. (Biology), San Diego State University, 1999 M.Sc. (Biology), Portland State University, 2005  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in The Faculty of Graduate Studies (Animal Science)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  August 2010  © Kristen Amy Walker, 2010  Abstract Marine mammal research often requires marking and tracking animals to collect longterm ecological data, but these procedures may cause pain. The aim of this thesis was to assess the behavioural and physiological effects of invasive marking and tracking techniques used on marine mammals. This thesis consists of 7 chapters, beginning with a general introduction (Ch. 1) and ending with a general discussion (Ch. 7). Chapter 2 reviews the literature on short- and long-term effects of marking and tagging, concluding that the preponderance of studies focus on injuries and behavioural changes and that no research prior to this thesis has assessed post-operative pain in marine mammals. Chapters 3 to 6 describe experiments designed to fill this gap by focussing on pain responses of endangered Steller sea lions to invasive marking (hot-iron branding) and tracking (implanting a tracking device via intra-abdominal surgery) procedures. Seven behaviours associated with postoperative pain were monitored for 3 d pre- up to 12 d post-surgery with the aim of describing behavioural responses after abdominal surgery (Ch. 3) and comparing the efficacy of two analgesic treatments (Ch. 4). In both studies sea lions spent more time with their back arched and standing, and spent less time lying on the ventral side and in locomotion after surgery, regardless of analgesic treatment. Chapter 5 described the behavioural responses of sea lions after hot-iron branding. In the 3 days after branding sea lions spent more time grooming their branded area, less time with pressure on their branded side, and less time in the pool and in locomotion. Chapter 6 assessed physiological (breathing and heart rate) and behavioural responses of anaesthetised sea lions during hot-iron branding. Sea lions had increased heart and breathing rate during and in the minutes after hot-iron branding. Behavioural responses during branding included trembling and head and shoulder movements. These findings ii  illustrate behavioural and physiological responses that can be applied to assessing pain in sea lions, and suggest that more effective analgesic protocols are required to mitigate pain responses after hot-iron branding and abdominal surgery.  iii  Table of contents Abstract................................................................................................................................... ii  Table of contents ................................................................................................................... iv  List of tables.......................................................................................................................... vii  List of figures ....................................................................................................................... viii  Acknowledgements ................................................................................................................ x  Co-authorship statement ...................................................................................................... xi  CHAPTER 1: General introduction..................................................................................... 1 1.1 1.2 1.3 1.4 1.5  Marking effects ............................................................................................... 1 Pain assessment ............................................................................................... 3 Research animals and facility .......................................................................... 4 Thesis objectives ............................................................................................. 5 References ....................................................................................................... 7  CHAPTER 2: The effects of marking and tagging on marine mammals: a review ...... 10 2.1 2.2 2.3  2.4  2.5 2.6 2.7  Introduction ................................................................................................... 10 Methods ......................................................................................................... 14 Results ........................................................................................................... 16 2.3.1 Types of marking studies involving marine mammals ..................... 16 2.3.2 Marking and tagging devices used .................................................... 25 Discussion and research recommendations ................................................... 37 2.4.1 What does the current research show? .............................................. 38 2.4.2 Where are the gaps in the literature? ................................................. 39 2.4.3 Where do we go from here? .............................................................. 40 2.4.4 Guiding principles for minimizing marking impacts ........................ 42 Conclusions ................................................................................................... 44 Acknowledgments ......................................................................................... 44 References ..................................................................................................... 45  iv  CHAPTER 3: Behavioural responses of juvenile Steller sea lions to abdominal surgery: developing an assessment of post-operative pain .............................................................. 53 3.1  3.2  3.3 3.4 3.5 3.6 3.7  Introduction ................................................................................................... 53 3.1.1 Pain assessment ................................................................................. 54 3.1.2 Aim ................................................................................................... 55 Methods ......................................................................................................... 56 3.2.1 Study design and animals .................................................................. 56 3.2.2 Study procedures ............................................................................... 57 3.2.3 Behavioural observations .................................................................. 58 3.2.4 Statistical analysis ............................................................................. 61 Results ........................................................................................................... 62 General discussion ........................................................................................ 68 Conclusion .................................................................................................... 71 Acknowledgments ......................................................................................... 71 References ..................................................................................................... 72  CHAPTER 4: The effects of two analgesic regimes on behaviour after abdominal surgery in Steller sea lions .................................................................................................. 75 4.1 4.2  4.3 4.4 4.5 4.6 4.7 4.8  Introduction ................................................................................................... 75 Methods ........................................................................................................ 77 4.2.1 Study design and animals .................................................................. 77 4.2.2 Study treatments ................................................................................ 78 4.2.3 Behavioural observations .................................................................. 79 4.2.4 Statistical analysis ............................................................................. 81 Results ........................................................................................................... 82 Discussion ..................................................................................................... 87 Conclusions ................................................................................................... 89 Conflict of interest ........................................................................................ 89 Acknowledgments ......................................................................................... 90 References ..................................................................................................... 91  CHAPTER 5: Behavioural responses of juvenile Steller sea lions to hot-iron branding ............................................................................................................................... 93 5.1 5.2  5.3 5.4  Introduction ................................................................................................... 93 Methods ......................................................................................................... 94 5.2.1 Study design and animals .................................................................. 94 5.2.2 Study procedures ............................................................................... 95 5.2.3 Behavioural observations .................................................................. 96 5.2.4 Statistical analysis ............................................................................. 98 Results ........................................................................................................... 99 General discussion ...................................................................................... 101  v  5.5 5.6 5.7  Conclusion .................................................................................................. 103 Acknowledgments ....................................................................................... 103 References ................................................................................................... 104  CHAPTER 6: Effects of hot-iron branding on heart rate, breathing rate and behaviour of anaesthetized Steller sea lions ...................................................................................... 106 6.1 6.2  6.3 6.4 6.5 6.6 6.7  Introduction .................................................................................................. 106 Methods ....................................................................................................... 108 6.2.1 Study animals .................................................................................. 108 6.2.2 Design ............................................................................................. 108 6.2.3 Physiological measures ................................................................... 110 6.2.4 Behavioural measures ...................................................................... 111 6.2.5 Statistical analyses .......................................................................... 111 Results ......................................................................................................... 112 Discussion ................................................................................................... 116 Conclusions ................................................................................................. 120 Acknowledgments ....................................................................................... 120 References ................................................................................................... 121  CHAPTER 7: General discussion..................................................................................... 124 7.1 7.2 7.3 7.4 7.5 7.6 7.7  Welfare concerns associated with abdominal surgery ................................. 125 7.1.1 Future abdominal surgery research ................................................. 126 Welfare concerns associated with hot-iron branding .................................. 128 7.2.1 Future branding research ................................................................. 129 Balancing animal welfare considerations with research goals ................... 130 Future directions in pain assessment research ............................................ 131 Conclusions ................................................................................................. 133 Personal recommendations ......................................................................... 134 References .................................................................................................... 136  Appendices .......................................................................................................................... 140 Appendix I – UBC Animal Care Certificates .......................................................... 140  vi  List of tables Table 2.1  Category A – Articles addressing the direct effects of marking. Category B – Articles testing the effectiveness of a marking device and mention the effects of the device ............................................................ 19  Table 3.1  Descriptions of behavioural activities recorded before and after LHX implant surgery ............................................................................................. 60  Table 3.2  Least square means and S.E.M. for the proportion of time sea lions (n = 9) spent displaying behaviours before and after LHX implant surgery. Means and S.E.M. are the arcsine square root transformed values. Backtransformed means are provided in parentheses. Specified contrasts pre- vs. post-surgery and pre- vs. late post-surgery P-values are presented and considered significant at P ≤ 0.05 .................................... 63  Table 4.1  Descriptions of instantaneous behaviours recorded before and after abdominal surgery ......................................................................................... 81  Table 5.1  Least square means and S.E.M. for the proportion of time sea lions (n = 11) spent engaged in six different behaviours before and after hotiron branding. Means and the S.E.M. are the arcsine square root transformed values (backtransformed  means are provided in  parentheses). Significant differences for specified contrasts (Pre-brand vs. Day 0, Day 1 and Day 2) are denoted by symbol * (P ≤ 0.05) .............. 100  vii  List of figures Figure 2.1  Number of studies published on marking effects between January 1980 - March 2010 ......................................................................................... 17  Figure 2.2  Effects of marking as reported in the 36 identified articles. Seventeen of the studies reported multiple effects ......................................................... 18  Figure 3.1  Least square means (± S.E.M.) for the proportion of time sea lions spent (a) displaying back arch behaviour while sitting upright and lying down while on land, (b) with pressure on the ventral side during periods of lying down or sitting upright while on land, and (c) in locomotion on both land and in the water. Means and S.E.M. are the arcsine square root transformed values. The x-axis represents time, presented as pre-surgery (average of 3 days before surgery), postsurgery (average of the 3 days immediately following surgery), and late post-surgery (average of Days 10-12 following surgery). Day effects on back arch, time on ventral side, and locomotion were significant at P < 0.001, P < 0.001 and P = 0.05, respectively ...................... 64  Figure 3.2  Least square means (± S.E.M.) for the proportion of time sea lions spent standing on land before and after LHX surgery. Means and S.E.M. are the arcsine square root transformed values. Open circles represent animals from Group 1 (n = 5) and closed squares represent animals from Group 2 (n = 4). The overall effect of day on standing  viii  behaviour was significant at P < 0.001. The interaction between line block and day on standing behaviour was significant at P = 0.047 .............. 66  Figure 4.1  Least square means and the S.E.M. for the proportion of time sea lions spent (a) alert, (b) locomotion, (c) lying down, (d) on ventral side, (e) stand, (f) back arch and (g) time in pool, before and after abdominal surgery. Means and S.E.M. are the arcsine square root transformed values. The x-axis represents time, presented as pre-surgery (average of 3 days before surgery), Day 0 (1st 24-h period after surgery), Day 13 (average of Days 1, 2, and 3 after surgery), and Days 4-6 (average of Days 4, 5, and 6 after surgery). Open circles represent animals administered carprofen (n = 5) and closed squares represent animals administered flunixin meglumine (n = 6) ..................................................... 83  Figure 6.1.  Instantaneous heart rates of 12 individual sea lions during four periods. Arrows represent beginning of a period; S – Sham branding, B – Branding, and P – Post-branding. Graphs begin with the 5 min Baseline period. Note, TJ 43 does not have a Post-brand period and TJ 51 had the Sham brand period occur before the Baseline period. Abscess wound cleaning is shown for TJ 50 and biopsy wound and blood draws for TJ 54 ............................................................................................ 113  Figure 6.2.  Average breathing rate (measured in breaths per minute) for sea lions during the 4 experimental periods. Data are presented as least square means ± S.E.M. ........................................................................................... 115  ix  Acknowledgments First and foremost, I would like to thank my supervisor Dr. Dan Weary for his continued guidance and encouragement through my PhD. He gave me the freedom to explore an area of research that I was passionate about and pushed me to think critically; he helped me develop tremendously as a researcher. I would also like to thank Dr. David Fraser for his guidance, clarity and for always sharing his boundless knowledge. I am grateful to Dr. Jo-Ann Mellish for enabling me to do my research, providing mentorship during my time in Alaska and for making me a part of the South Beach Team. I am also grateful to my other committee members: Dr. Martin Haulena for his expertise and advice in the area of marine mammal anaesthesia and analgesia and to Dr. Andrew Trites for his guidance throughout my PhD. An additional thank you goes to Dr. Nina von Keyserlingk for her guidance and for always having time to talk with me – whether about my work or my growth as a researcher. I would like to thank the veterinary and husbandry staff, especially Dr. Pam Tuomi, at the Alaska SeaLife Center for their support with my project. Their constant commitment to the animals is what made my research a success. And of course, a special thank you goes to the TJs for unwillingly taking part in our research; and to my Alaskan friends who provided me with the true Alaskan experience – a year just was simply not long enough. My experience in the Animal Welfare Program has an unforgettable one. The program provided such an enriching and supportive environment. The dedication and passion of Drs. Weary, Fraser and von Keyserlingk to their student’s growth and education is inspirational. I am grateful to all the students in the Animal Welfare Program for their support over the years. In particular, I would like to thank my friend and fellow wildlife enthusiast Liv, for always being there for me; Katy, for her enthusiasm and dedication to our program, and for always being willing to help and provide advice; and Nuria, for her continued statistical support, friendship and guidance. A special thank you goes to my friends Tiffany and Nikki for always believing in and being there for me, no matter how far the distance; and to Dr. Debbie Duffield for opening the door for me years ago, your belief in me is what enabled me to pursue my passions. Lastly, I could not have done this journey without my family’s support and unconditional love. I would like to thank my Mom, Dad, and big sis’ Jennifer – your support and encouragement is what made the pursuit of my dreams possible; and Simba for his companionship and never-ending affection.  x  Co-authorship statement Study design, performance, statistical analysis, interpretation and write-up for all chapters in this dissertation were performed by Kristen Amy Walker, under the supervision of Dr. Daniel Weary. The following co-authors were involved in the design, interpretation and preparation of the manuscripts included in this dissertation: Drs. Andrew Trites, Martin Haulena and Daniel Weary for Chapter 2; Drs. Markus Horning, Jo-Ann Mellish and Daniel Weary for Chapters 3 and 4; and Drs. Jo-Ann Mellish and Daniel Weary for Chapters 5 and 6.  xi  CHAPTER 1: General introduction 1.1 Marking effects Marine mammals are vital to marine ecosystems, with great potential economic impact on important fisheries areas such as the Bering Sea. Although declines in several marine mammal populations over the past few decades have been documented, the decline in the Western United States population of Steller sea lions (Eumetopias jubatus) has received particular research focus. This population of Steller sea lions is currently listed as endangered under the U.S. Endangered Species Act of 1973 and depleted under the Marine Mammal Protection Act of 1972. The National Marine Fisheries Service (NMFS) has formulated a Steller Sea Lion Recovery Plan. A primary objective of the plan is to develop methods that minimize the impacts of research programs, to prevent any interference with the time to recovery of the population (NMFS, 2008). To understand the sea lion decline and the cause of their disappearance, life history traits need to be studied but the collection of such data typically requires that individuals be handled and marked. Methods used to mark marine mammals include ear and flipper tags, external satellite and radio transmitters, hot and cold branding, and surgically placed transmitters (Wells, 2002). Accurate identification of individuals over time allows for data collection on social behaviour, survival, reproduction, home-range use and resource selection (Murray and Fuller, 2000). Hot-iron branding has been a successful way to permanently mark sea lions and other marine mammals (Wells, 2002), but has also proven controversial. For example, concerns raised over hot-iron branding were sufficient to result in the revocation of all Steller sea lion  1  research permits for a period of over a year (Dalton, 2005) and have resulted in indefinite suspensions of hot-iron branding for elephant seals at Macquarie Island and Hooker’s sea lions in New Zealand (Beausoleil and Mellor, 2007; McMahon et al., 2007). Efforts have been made recently to gain a more comprehensive understanding of the effects of marking techniques on animals both to ensure future permitting of tracking studies (e.g., Mellish et al., 2007; Horning et al., 2008) and to recognize the potential confounds that may be introduced from marking animals (i.e., are data collected on marked animals representative of the unmarked population). Advances in technology have allowed researchers to collect data on animal mortality, maternal care, dive behaviour, and physiology including heart rate, core and stomach temperatures. For example, tracking devices such as the Life History Transmitter (LHX tag) give researchers the ability to collect life-long data in pinnipeds (Horning and Hill, 2005). However, with the development of new technology there is a need to determine how this affects the animal. Data collected in field research are important for wildlife management and conservation; however, the research may come at a cost to the animal and these impacts should be assessed. Studies on non-marine mammal species have shown that markings can affect growth rates, cause pain and distress, influence an animal’s thermoregulatory abilities, cause drag, and even influence mate choice (Murray and Fuller, 2000). Marine mammals are subjected to marking procedures that may interfere with the animal’s natural behaviours and cause pain, but there are few data available to evaluate such marking effects. As argued by Baker and Johanos (2002), previous researchers may have ignored the topic because unmarked animals are difficult to follow in the wild, or simply because researchers felt the  2  effects of marking were negligible. To date, research on marker effects has primarily focused on short-term effects such as injuries and behavioural changes, and long-term effects on survival (see Chapter 2 for review). Research has not considered chronic effects of marking and tagging on reproduction and health. Moreover, no study has assessed the pain responses of marine mammals to invasive marking or tagging procedures.  1.2 Pain assessment As defined by the International Association for the Study of Pain, pain is “an unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage” (IASP, 1994). Changes in general body functioning (e.g., decreased food and water consumption), physiological measures (e.g., heart and breathing rate), and behavioural measures have been used to assess pain in animals (Weary et al., 2006). Vocalizations have also been used as indicators of pain and distress, such as during castration in pigs or branding in cattle (Taylor et al., 2001; Watts and Stookey, 1999). Pain measures can be validated by observing responses with and without a paincausing condition, and with and without medications such as analgesics that are known to be effective at treating pain (Dobromylskyj et al., 2000). However, the inclusion in experiments of a control group of animals administered a noxious stimulus without the administration of analgesia raises ethical concerns. Three main classes of behaviours can be used in assessing pain: (1) pain-specific behaviours, (2) decline in the frequency or magnitude of particular behaviours, and (3) choice or preference behaviours (Weary et al., 2006). Behavioural responses will vary among species. Pain-specific behaviours may occur during to the procedure itself or may be  3  witnessed for days to weeks after a painful procedure due to prolonged tissue damage and inflammation. Behavioural responses to pain have been studied in a range of species including in cattle during dehorning (Morisse et al., 1995; Faulkner and Weary, 2000; Vickers et al., 2005), in fish during noxious stimulation (Sneddon, 2003), in rats after surgery (Roughan and Flecknell 2001, 2003, 2004), and in lambs during castration and tail docking (Molony et al., 2002). Pain can also be assessed using physiological measures including activation of the stress response systems. Responses include changes in heart and breathing rates, hormone concentrations (including glucocorticoids and catecholamines) and blood pressure. Examples of studies looking at physiological responses to pain include assessing cortisol levels after castration (calves; Stafford et al., 2002), heart and breathing rates during disbudding (goats; Alvarez et al., 2009), heart rate variability due to laminitis (horses; Rietmann et al., 2004) and eye temperature and heart rate variability during disbudding (calves; Stewart et al., 2008).  1.3 Research animals and facility It has been suggested that assessments of marking effects should be conducted in captive, controlled environments using appropriate controls (Baker and Johanos, 2002). The Transient Juvenile Steller Sea Lion Project at the Alaska SeaLife Center (ASLC) in Seward, AK, provides an opportunity to assess behavioural and physiological parameters in a controlled setting (Mellish et al., 2006). Each year up to 24 juvenile Steller sea lions, between 16 and 36 months of age, are captured from the Prince William Sound, AK. These animals are transported to a specialized quarantine facility at ASLC for short-term projects  4  (up to 3 months). No more than 6 individuals are brought to the facility at one time. The Transient project utilizes the sea lions for a variety of observational and experimental studies of health, body condition, nutrition and validation of new tracking and assessment technologies. During captivity sea lions are subjected to numerous handling events to assess health status, body condition and stress responses. In addition, prior to release back to their natural habitat, all individuals are branded with a hot iron and most receive an LHX tag implant. Animals are also outfitted with an external satellite tag for post-release monitoring. All animals described in this thesis were captured, branded and implanted as part of the requirements for other research projects falling under the Transient Juvenile Steller Sea Lion Project; no animals were branded or underwent abdominal surgery for the purposes of this thesis, and no additional handling was required for the behavioural and physiological observations I conducted.  1.4 Thesis objectives There is a lack of knowledge on the effects of marking and tracking devices on marine mammals. The objective of Chapter 2 was to identify gaps in the marine mammal literature by completing a review of published peer-reviewed articles. In this review, I provided a critical assessment of the studies reporting on marking and tagging effects on marine mammals. One of the major conclusions was that no published research on marine mammals has specifically assessed pain and distress associated with marking or tagging. For this reason, the overall objective of my dissertation was to assess the pain responses in Steller sea lions after hot-iron branding and abdominal surgery for telemetry device implantation.  5  To address this overall objective I applied: 1) behavioural techniques to assess the pain responses of sea lions witnessed in the days after abdominal surgery (Chapters 3 and 4) and hot-iron branding (Chapter 5), and 2) a combination of behavioural and physiological techniques to assess the immediate pain responses of sea lions to hot-iron branding (Chapter 6).  In summary, the aim of my thesis was to provide a scientific basis for the evaluation of different marking techniques used in sea lions and to provide information on how to mitigate the potential negative effects on the animal.  6  1.5 References Alvarez, L., Nava, A.R., Ramirez, A., Ramirez, E., Gutierrez, J. 2009. Physiological and behavioural alterations in disbudded goat kids with and without local anaesthesia. Applied Animal Behaviour Science 117: 190-196. Baker, J. D., Johanos, T.C. 2002. Effects of research handling on the endangered Hawaiian Monk seal. Marine Mammal Science 18: 500-512. Beausoleil, N.J., Mellor, D.J. 2007. Investigator responsibilities and animal welfare issues raised by hot branding of pinnipeds. Australian Veterinary Journal 85: 484-485. Dalton, R. 2005. Animal-rights group sues over ‘disturbing’ work on sea lions. Nature 436: 315. Dobromylskyj, P., Flecknell, P.A., Lascelles, B.D., Pascoe, P.J., Taylor, P., WatermanPearson, A. 2000. Management of postoperative and other acute pain. Pages 81-145 in Pain Management in Animals. Flecknell, P., Waterman-Pearson, A. (Eds.), Harcourt Publishers Limited, London, UK. Faulkner, P.M., Weary, D.M. 2000. Reducing pain after dehorning in dairy calves. Journal of Dairy Science 83: 2037–2041. Horning, M., Hill, R.D. 2005. Designing an archival satellite transmitter for life-long deployments on oceanic vertebrates: the life history transmitter. IEEE Journal of Oceanic Engineering 30: 807-817. Horning, M., Haulena, M., Tuomi, P.A., Mellish, J.E. 2008. Intraperitoneal implantation of life-long telemetry transmitters in otariids. BMC Veterinary Research 4: 51 doi:10.1186/1746-6148-4-51. International Association for the Study of Pain (IASP) 1994. Task Force on Taxonomy, Pages 209-214 in Classification of Chronic Pain, 2nd Edition. Merskey, H., Bogduk, N. (Eds), International Association for the Study of Pain, Seattle, WA. McMahon, C. R., Bradshaw, C. J. A., Hays, G. C. 2007. Applying the heat to research techniques for species conservation. Conservation Biology 21: 271-273. Mellish, J., Calkins, D., Christen, D., Horning, M., Rea, L., Atkinson, S. 2006. Temporary captivity as a research tool: comprehensive study of wild pinnipeds under controlled conditions. Aquatic Mammals 32: 58-65. Mellish, J., Hennen, D., Thomton, J., Petrauskas, L., Atkinson, S., Calkins, D. 2007. Permanent marking in an endangered species: physiological response to hot branding in Steller sea lions (Eumetopias jubatus). Wildlife Research 34: 1-6.  7  Molony, V., Kent, J.E., McKendrick, I.J. 2002. Validation of a method for assessment of an acute pain in lambs. Applied Animal Behaviour Science 76: 215-238. Morisse, J.P., Cotte, J.P., Huonnic, D. 1995. Effect of dehorning on behaviour and plasma cortisol responses in young calves. Applied Animal Behaviour Science 43: 239-247. Murray, D.L.,Fuller, M.R. 2000. A critical review of the effects of marking on the biology of vertebrates. Pages 15-64 in Research Techniques in Animal Ecology. Boitani, L., Fuller, T. (Eds.), Columbia University Press, New York, NY. National Marine Fisheries Service, NMFS. 2008. Recovery Plan for the Steller Sea Lion (Eumetopias jubatus). Revision. National Marine Fisheries Service, Silver Spring, MD. 325 pgs. Rietmann, T.R., Stauffacher, M., Bernasconi, P., Auer, J.A., Weishaupt, M.A. 2004. The association between heart rate, heart rate variability, endocrine and behavioural pain measures in horses suffering from laminitis. Journal of Veterinary Medicine 51: 218-225. Roughan, J.V., Flecknell, P.A. 2001. Behavioural effects of laparotomy and analgesic effects of ketoprofen and carprofen in rats. Pain 90: 65-74. Roughan, J.V., Flecknell, P.A. 2003. Evaluation of a short duration behaviour-based postoperative pain scoring system in rats. European Journal of Pain 7: 397-406. Roughan, J.V., Flecknell, P.A. 2004. Behaviour-based assessment of the duration of laparotomy- induced abdominal pain and the analgesic effects of carprofen and buprenorphine in rats. Behavioural Pharmacology 15: 461-472. Sneddon, L. 2003. The evidence for pain in fish: the use of morphine as an analgesic. Applied Animal Behaviour Science 83: 153-162. Stafford, K.J., Mellor, D.J., Todd, S.E., Bruce, R.A., Ward, R.N. 2002. Effects of local anaesthesia or local anaesthesia plus a non-steroidal anti-inflammatory drug on the acute cortisol response of calves to five different methods of castration. Research in Veterinary Science 73: 61-70. Stewart, M., Stafford, K.J., Dowling, S.K., Schaefer, A.L., Webster, J.R. 2008. Eye temperature and heart rate variability of calves disbudded with or without local anaesthetic. Physiology & Behavior 93: 789-797. Taylor, A.A., Weary, D.M., Lessard, M. Braithwaite, L. 2001. Behavioural responses of piglets to castration: the effect of piglet age. Applied Animal Behaviour Science 73: 35-43. Vickers, K.J., Neil, L., Kiehlbauch, L.M., Weary, D.M. 2005. Calf response to caustic paste and hot-iron dehorning using sedation with and without local anesthetic. Journal of Dairy Science 88: 1454-1459.  8  Watts, J.M., Stookey, J.M. 1999. Effects of restraint and branding on rates and acoustic parameters of vocalization in beef cattle. Applied Animal Behaviour Science 62: 125-135. Weary, D.M., Neil, L., Flower, F.C., Fraser, D. 2006. Identifying and preventing pain in animals. Applied Animal Behaviour Science 100: 64-76. Wells, R.S. 2002. Identification methods. Pages 601-608 in Encyclopedia of Marine Mammals. Perrin, W.F., Wursig, B. & Thewissen, J.G.M. (Eds). Academic Press, London, UK.  9  CHAPTER 2: The effects of marking and tagging on marine mammals: a review 1 2.1 Introduction Wildlife research often requires marking or tagging animals to identify individuals and obtain data on social behavior, survival, reproduction, home range use, and resource selection (Merrick et al. 1996; Murray and Fuller 2000). Methods for long-term identification include brands, implanted coded tags, scarring (e.g., toe or fin clipping), and external numbered tags attached to ears, legs or flippers (Merrick et al. 1994; Horning et al. 1999; Murray and Fuller 2000; Wells 2002; Lander et al. 2005). Methods used for short-term identification include paint, hair dye and electronic instruments glued to fur and feathers, or carried around necks and legs (e.g., VHF radio tags or satellite tracking tags). Some marking and tagging techniques have been used for centuries (e.g., branding), and others have been used for less than a decade (e.g., internally implanted satellite tags). Data collected from marked animals are important for scientists, but the marking or tagging techniques may come at a cost to the animal. Some changes in behavior or physiology due to marking may be sufficiently severe as to also affect our ability to interpret data from animals marked in this way. Marking can have adverse effects on the individual animal, as well as on populations and interactions between species. Studies on a range of non-marine mammal species have shown that markings can decrease growth rate, interfere with natural behavior, cause pain and distress, and reduce survival and reproduction (Pavone  1  A version of this chapter has been submitted for publication. Walker, K.A., Trites, A., Haulena M. and Weary, D.M. 2010. The effects of marking and tagging on marine mammals: a review. 10  and Boonstra 1985; Pietz et al. 1993; Schwartzkopf-Genswein et al. 1997c; Swenson et al. 1999). Marking can affect reproduction and mate choice; for example, zebra finches (Poephila guttata) fitted with colored plastic leg bands showed mating preferences for particular colors (red, pink, and black bands) while avoiding mating with other birds fitted with light-blue or light-green bands (Burley et al. 1981). Pavone and Boonstra (1985) provide an example of decreased survival by showing that the commonly used marking method of toe-clipping decreased the overall life span of meadow voles (Microtus pennsylvanicus). Modifications to devices or the improper placement of devices can also decrease survival. For example, moose (Alces alces) calves fitted with ear transmitters had lower survival than calves fitted with only ear-tags (Swenson et al. 1999). A mark or tag may also hinder an animal’s ability to escape predators if it inhibits their mobility or if the presence of the marker makes the animal more visible. For instance, Webster and Brooks (1980) found reduced survival in meadow voles in the winter, with animals carrying radio transmitters being more susceptible to predation than those without radio transmitters. Injury and disease can occur in response to marking. For example, hot-iron branding and toe-clipping leave exposed tissue susceptible to infection. The marker can cause abrasions, entanglement, or compression of tissues. Ear or flipper tags can be ripped out and leave an open wound that can become infected (Nietfeld et al. 1994; Wells 2002). Marking devices placed subcutaneously may be rejected from the animal’s body and cause tissue reactions or infection (Lander et al. 2005; Green et al. 2009). Surgically implanted radio transmitters have been found to cause infection or mortality as a result of the surgical procedure or due to intestinal obstruction (Eagle et al. 1984; Guynn et al. 1987; Hernandez-  11  Divers et al. 2001). External radio devices attached by epoxy-glue can leave the skin irritated and raw. Arm, neck, or leg bands and radio collars can also cause abrasions, open wounds, and restrict limb circulation and movement (Eagle et al. 1984; Nietfeld et al. 1994; Baker et al. 2001). Markers may also impede the animal’s ability to perform natural behaviors such as locomotion, feeding or escaping from predators. The inability to perform natural behaviors may lead to frustration, injury and disease, or death. For example, Pietz et al. (1993) found that the placement of radio transmitters on mallard ducks (Anas platyrhynchos) interfered with time spent feeding and caused overall weight loss. The weight or presence of a device may also increase energy expenditure. For example, penguins (Pygoscelis sp.) and green turtles (Chelonia mydas) fitted with external data loggers and transmitters experienced drag, which decreased swimming speeds and increased energy expenditure (Bannasch et al. 1994; Watson and Granger 1998). If the marker disrupts the plumage or pelage, thermoregulatory ability may be reduced resulting in heat loss or mortality, as witnessed in mallard ducks (Bakken et al. 1996) and sea otters (Hatfield and Rathbun 1996). Animals may further be affected by the procedures required to mark or tag them (i.e., capture, handling and restraint; Mellor et al. 2004), which could cause animals to experience fear or anxiety. Human handling can create fear in animals (Hemsworth 2004); for example, aversive handling of cows during milking led to lower milk yield and increased heart rate (Rushen et al. 1999). Capture myopathy is a metabolic disease involving muscle necrosis, metabolic acidosis, and myoglobinuria that is often associated with capture and handling and can occur in a variety of birds and mammals (Spraker 1993; Curry 1999; Herraez et al.  12  2007). Capture myopathy may result in death days or weeks after the exertion (Paterson 2007). Another concern is the pain associated with the marking procedure and the subsequent healing process. For example, acute pain responses of cattle (Bos taurus) have been studied during hot-iron and freeze branding. Compared to freeze branding, hot-iron branding results in greater escape-avoidance reactions (Lay et al. 1992a), a greater incidence of behaviors such as tail-flicking, kicking, and falling, and more prolonged physiological responses including elevated heart rate and plasma concentrations of cortisol and epinephrine (Lay et al. 1992a, b; Schwartzkopf-Genswein et al. 1997a, b, c, 1998). Researchers tend to choose marking and tagging methods they believe will minimize abnormal behavior or do not negatively affect survival. However, this decision is often based on intuition rather than experimental data (Murray and Fuller 2000). For example, Smith et al. (1973) stated that hot-iron branding of ringed seals (Phoca hispida) “caused little apparent distress to the animals”, but did not collect measures of distress to substantiate their statement. Similarly, Williams and Siniff (1983) wrote that “the advantage of the surgically implanted devices [in sea otters (Enhydra lutris)] is that they offer no impediment physically or behaviorally”, but did not provide behavioral data to support their claim. Such unsupported statements about the lack of marking effects on study animals may reflect the difficulty of following control (unmarked) animals in the wild, or may simply reflect the feeling of researchers that these effects are negligible (Baker and Johanos 2002). Marine mammal research is increasingly relying on marking and tagging individual animals to answer questions about population dynamics (e.g., birth and survival rates), behavioral ecology (e.g., foraging, mate choice) and physiology (e.g., energy requirements)  13  (e.g., Hedd et al. 1995; Merrick and Loughlin 1997; Andrews et al. 2002; Maniscalco et al. 2006). However, few studies have directly addressed the effects of marking on marine mammals, and many appear to not fully appreciate how their data may be compromised by the behavioral and physiological effects of marking or tagging. With this in mind we reviewed 30 yr of research to assess what is known about the effects of different marking and tagging techniques on marine mammals.  2.2 Methods We evaluated journal articles pertaining to marking marine mammals that were published in peer-reviewed journals. We have chosen not to include the grey literature (e.g., governmental reports or conference abstracts) in this review due to inconsistencies in quality that the peer-review process provides. We began our search for relevant articles with searches in Web of Science, IngentaConnect and a Google Scholar search using the terms ‘marking’, ‘tagging’, or ‘transmitter’ in combination with the terms ‘effect(s)’, ‘evaluation’, or ‘response’. Using the articles identified by these searches we then scanned the literature backwards (using the papers cited in these articles) and forwards (by seeing who has later cited these articles) in time, repeating this process every time a relevant article was identified. We evaluated journal articles published between January 1980 and March 2010. This time frame was chosen due to the online availability and reliability of searching services since 1980. We restricted our search to articles that used markers that uniquely identified individual animals, and excluded devices such as the Crittercam (National Geographic Television, Washington DC, U.S.A.) and video cameras that do not provide a uniquely identifiable mark.  14  For the ease of discussion, we will use the term ‘marking’ to include the use of marking devices such as paint or hot-iron brands, radio and satellite telemetry devices, as well as data loggers. Studies were grouped into five categories: (A) studies designed to show how a particular marker affects the animal (e.g., assessing wound healing after hot-iron branding), (B) studies that tested the feasibility of marking and reported incidentally on the effects on the animal (e.g., successful placement of PIT tags in sea otters), (C) studies that used markers to study the behavior of animals and report incidentally on marking effects (e.g., movement data collected by radio telemetry devices), (D) studies that described capture, handling and marking techniques for a particular species (e.g., beluga capture and handling techniques) and (E) studies that described the frequency of tag loss and its effects on population data. We focused on articles that assess the direct effect of marking and tagging devices and therefore fall into categories (A) and (B). Readers interested in capture and handling effects or tag loss are refereed to studies such as Curry (1999), McMahon et al. (2005), and Testa and Rothery (1992). Studies falling within categories (A) and (B) were then grouped by marking or tagging method: 1) external tracking or telemetry devices, 2) implanted tags for marking, 3) hot- and cold-iron branding, and 4) visual tags. These grouping were chosen based on how the device was affixed to the animal and the tissue manipulation/destruction involved. Specifically, external devices cause minimal to moderate tissue manipulation/destruction; implanted tags cause minimal to severe tissue manipulation; branding causes tissue destruction; and visual tags involve minimal tissue manipulation. These categories were based on guidelines in CCAC (2003). Within each marking type we examined five types of  15  effects that marking devices may have on animals: (1) behavior (e.g., changes in swimming behaviors, haul out behavior, group structure, migration, trip length), (2) physiology (e.g., changes in heart rate, hematology and serum chemistry, cortisol levels, heat flux), (3) injury and disease (e.g., wound healing, tissue damage and histological changes), (4) survival, and (5) reproduction and growth.  2.3 Results 2.3.1 Types of marking studies involving marine mammals We identified 36 studies that addressed the effects of marking on marine mammals; 19 of these were published since 2000 (Fig. 2.1). Most of the 36 studies focused on behavioral changes and the injuries caused by the placement of the markers, and 17 of the 36 studies considered multiple effects (Fig. 2.2). The majority of studies that addressed behavior and injury found effects, but the responses varied by marking device and species studied. Most studies on survival reported no lethal effect of the marking device, but studies looking at physiological changes all reported measurable effects. No studies reported significant effects on reproduction or growth. Of the 36 studies, 21 were in Category A (13 species; Table 2.1) and 15 in Category B (13 species; Table 2.1).  16  20 16 12 8 4 0 1980-1989  1990-1999  2000-2010  Year Figure 2.1 Number of studies published on marking effects between January 1980- March 2010.  17  20  Number of studies  16 12 8 4 0 Behavior  Physiology  Injury & Disease  Survival  Reproduction & Growth  Figure 2.2 Effects of marking as reported in the 36 identified articles. Seventeen of the studies reported multiple effects.  18  Table 2.1 The 36 articles that address the effects of marking on marine mammals: Category A represents articles addressing the direct effects of marking and Category B represents articles testing the effectiveness of a marking device and mention the effects of the device. Category A  Author  Irvine et al. 1982  Species  Atlantic bottlenose dolphin  Number of animals  3  Measured Effects4  Duration of reported effects5  bolt  Mod  B*, I*  L  dorsal fin  single bolt  Mod  I*  L  double bolts  Mod  I*  L  Perm  dorsal fin both sides of dorsal fin; on body below fin  irons cooled in dry ice  Sev  Semi  trailing edge of dorsal fin  Level of tissue  Marking1  Duration of marking2  7 single, 3 double bolt  RT  Semi  dorsal fin  16  visual tag  Semi  19  visual tag  Semi  47  FB  53  roto tags  17  spaghetti tags  Henderson and Johanos 1988  Hawaiian Monk seal  Trites 1991  Northern fur seal  Watkins and Tyack 1991 Walker and Boveng 1995  Sperm whale Antarctic fur seal  105  FT monel metal sonar transponder and RT TDR and RT  Schneider and Baird 1998  Bottlenose dolphin  10  Hanson and Baird 1998  Dall's porpoise  13  13  200000  2  plastic FT  Placement  Method of attachment  Mod  Temp  Semi  rear flipper between 4th and 5th digit  Semi  front flipper  manipulated  metal screw  I*  U  Injury type bolts tore fin leaving necrotic discolored wounds  Stats6  Captive or wild  N  W  fin damage skin abrasions, bolt wounds  N  W  N  W  N  W  N  W  N  W  Y  W  fin damage entry wounds tag tore out tearing tissue  Min  I*  L  Mod  S, B*, I*  L  Min  R  NA  Y  W  implanted in skin  Mod  B  NA  N  W  Semi  above dorsal hump pelage of midback  epoxy glued  Min  B*  Y  W  RT (VHF) and TDR  Temp/ Semi  dorsal side  suction cup  Min  B*  N  W  RT  Temp/ Semi  dorsal side  suction cup  Min  B*  Y S – only report acute response S – only report acute response  N  W  Perm  19  Hooker et al. 2001  Northern bottlenose whale  47 56  Baker and Johanos 2002  Geersten et al. 2004  Hawaiian Monk seal  Harbor porpoise  RT (VHF) and TDR satellite TDR  Temp/ Semi  epoxy glued  Min  S, B  NA  Y  W  epoxy glued  Min  S, B  NA  Y  W  epoxy glued  Min  S, B  NA  Y  W  Min  S, B  NA  Y  W  B*, P*, I*  S – tags only attached for 7 d  reddish exudate at attachment site  Y  C  brand wound  Y  W  Y  W  Y  W  TDR  Semi  5  GPS data logger  Semi  dorsal pelage  437  plastic flipper tags  Semi  rear flipper  Semi  dorsal fin  Sev  I*  mid region of back  Sev  I*  L  Semi  dorsal fin  steel borers  Mod  S, R, I*  Y  Perm  each side of caudodorsal flank  hot-iron; liquid nitrogen or dry ice cooled iron  Sev  S  Y  W  Perm  ventrocauda l abdominal cavity  intraperitoneal cavity, freefloating  Sev  B, P*  Y  Semi C  Perm  left shoulder/ flank  hot-iron  Sev  P*  NA L – return to baseline at 6wk postimplant L –return to baseline 7-8wk postbrand  Y  C  dorsal side  velcro patches epoxy glued to pelage  Min  P*  U  Y  C  2466  HB  Perm  right and left flank  Daoust et al. 2006  Harbor seal  306  HB and FB  Perm  Martin et al. 2006  Amazon river dolphin  51  RT  McMahon et al. 2006  Southern elephant seal  Mellish et al. 2007a  Mellish et al. 2007b  McCafferty et al. 2007  Steller sea lion  Steller sea lion  grey seal  6  7  2  LHX  HB heart rate transmitter and recorder  Mod  W  hot-iron hot-iron; liquid nitrogen cooled iron  Southern elephant seal  HB and FB  3 holes bored in fin, fixed with pins and nuts  Y  Y – most healed within a year  van den Hoff et al. 2004  14050  B*  Semi  8  satellite and RT (VHF)  Min  dorsal side dorsal pelage dorsal pelage  1  suction cup  S – only report acute response  Semi  20  brand wound holes and wounds where pins were  McMahon et al. 2008  Southern elephant seal  124  TDR, RT (VHF),and platform transmitter terminals  Hasting et al. 2009  Steller sea lion  366  HB, FT  Walker et al. 2009  Walker et al. 2010  Steller sea lion  Steller sea lion  Semi  dorsal side  epoxy glued to pelage  Min  S, R  NA  Y  W  Perm  dorsal side, fore flippers  hot-iron  Sev  S  Y  W  Perm  ventrocauda l abdominal cavity  intraperitoneal cavity, freefloating  Sev  B*  NA L – did not monitor past 12 d  Y  Semi C  HB  Perm  left shoulder/ flank  B*  S – did not monitor past 3 d  Y  Semi C  Number of animals  Marking1  Duration of Marking2  manipulate d3  Effects addressed4  Duration reported of effects5  Stats6  Captive or wild  3  RT  Semi  neck  collar  Min  S*, I*  L  N  W  6  RT  Semi  ankle  ankle bracelet  Min  S*, I*  L  N  both  144  RT  Semi  rear flipper  steel bolts and nuts  Mod  S, B*, I*  L  N  W  Perm  ventrally, below umbilicus  Mod  S*, B*, I*  U  N  W  Perm  ventrally, below umbilicus  Sev  S (?)  U  N  W  N  W  9  11  LHX  hot-iron  Category B  Author  Garshelis and Siniff 1983  Species  Sea otter  10  5  Mate and Harvey 1983  Gray whale  14  RT  RT  RT  Perm  Sev  Level of tissue Placement  1m behind blowhole  Method of attachment  subcutaneous intraperitoneal sutured to abdominal musculature  implanted in blubber  21  Mod  B, I*  L  Injury type neck constriction swollen ankle broken digits, slits in webbing removed sutures, exposed device  swelling at entry site 13 days after tag attachment  Williams and Siniff 1983  Griben 1984  Thomas et al. 1987  Ralls et al. 1989  Goodyear 1993  Hatfield and Rathbun 1996  Wright et al. 1998  Mulcahy and Garner 1999  Sea otter  Northern Fur Seal  Sea otter  5  Mod  S*, I*  S  intraperitoneal cavity, sutured to peritoneum  Sev  S  U  N  W  N  W  N  W  N  C  N  W  RT  Perm  2535  FP  Temp  head and back  pelage  Min  I, B  NA  Perm  base of neck top of shoulders  subcutaneous  Mod  I  NA  No injury noted no migration, infection or tissue damage noted  Perm  ventrally, below umbilicus  intraperitoneal cavity, free floating  Sev  S, I  NA  No injury noted  Perm  back  implanted in blubber  Mod  B*  S  N  W  Perm  back  implanted in blubber  Mod  B*  S  N  W  Y  W  N  C  N  W  6  North Atlantic right whale  13  Humpback whale  4  Polar Bear  subcutaneous  10  40  Manatee  Perm  ventrally, below umbilicus  Sea otter  Sea otter  RT  ventrally, below umbilicus  contusion abd. wall, hemorrage subcut., infection, suture removal  75  PIT tags  RT sonic transmitter; VHF radio transmitter sonic transmitter; VHF radio transmitter  RT  5  PIT tags  7  satellite transmitter  Semi  hind flipper  Min  S(?), B*, I*  U  Perm  middle of freeze brand  subcutaneous  Mod  I*  L  Perm  dorsal cervical area  subcutaneous  Mod  I*  L  22  tags tore flipper webbing injection sites raised and hard, then became flat. sutures broken leaving gaps at exudate drainage  Galimberti et al. 2000  Southern elephant seal  Blomqvist and Amundin 2004  Bottlenose dolphin  Lander et al. 2005  Harbor seal  California sea lion  510  PIT tags  Perm  rear flipper  subcutaneous  Mod  S, I  2  Acoustic tag  Temp/ Semi  dorsal fin  suction cup  Min  B*  15  RT encased in resin or wax  4  LHX  Perm  Perm  left dorsal thorax ventrocaud al abdominal cavity  subcutaneous  Mod  S(?), P*, I*  NA U – tags were only temporar y attached L – tissue reaction resin RT; S- wax RT  intraperitoneal cavity, freefloating  Sev  S, B*, I*  S  intraperitoneal cavity, freefloating  Sev  S, B*, I*  S  Horning et al. 2008  Steller sea lion  California sea lion  15  LHX  3  heart rate data logger  Perm  ventrocaud al abdominal cavity right thoracic and lumbar flank  Perm  right thoracic and lumbar flank  Perm  subcutaneous  Mod  I, P*  S  I*, P*  L– removed implant due to rejection  Green et al. 2009  Northern elephant seal  3  heart rate data logger  subcutaneous  1  Mod  no tissue reaction observed  wound discharge and opening minimal swelling at incision site minimal swelling at incision site; mild clear discharge noted in 2 animals minimal swelling and no exudate presented with swelling and pus and mucus exudate  Y  W  Y  C  N  both  N  Semi C  N  Semi C  N  Semi C rehab  N  Semi C rehab  Marking method - fluorescent paste (FP), radio transmitter (RT), flipper tags (FT), time depth recorder (TDR), passive integrated transponder (PIT), hotiron branding (HB), freeze-iron branding (FB), bleach mark (B), Paint mark (P), Life History Transmitter (LHX). 2 Duration of marking – temporary (Temp), semi-permanent (Semi), permanent (Perm) based on classification proposed by Mellor et al. (2004). 3 Level of tissue manipulated – minimal (Min; tissue manipulation, but no tissue destruction), moderate (Mod; moderate tissue manipulation and or tissue destruction), severe (Sev; severe tissue manipulation or destruction; categories formed based on guidelines in CCAC, 2003). 4 Measured marking effects included: Behavioral (B), Physiological (P), Injury and Disease (I), Survival (S), and Reproduction and Growth (R). An * indicates that the authors reported an effect. 5 Duration of reported effects – short-term (S; less than a week), long-term (L; weeks to months), years (Y), unknown (U), or not applicable (NA) due to no measurable effects. 6 Stats: No the study did not report the use of statistical analyses (N), or yes the study did report some form of statistical analysis in the article (Y).  23  2.3.1.1.Category A – Studies designed to show how marking directly affects the animal The 21 studies that investigated how marking directly affects marine mammals focused primarily on behavioral effects, followed by survival effects and injuries. Multiple effects were investigated in seven of the 21 studies. All articles assessing physiological changes and tissue injury reported significant effects (n = 8); however, none of the studies reported significant effects of marking on survival (n = 6), reproduction or growth (n = 3). Externally attached radio transmitters and time-depth recorders were the most commonly studied marker types, and were affixed to the animals in different ways. The effects of hotand cold-iron branding and visual tags were also studied directly. Pinnipeds were the most commonly studied group of marked marine mammals (7 of the 13 species) — particularly Southern elephant seals (Mirounga leonina) and Steller sea lions (Eumetopias jubatus).  2.3.1.2 Category B – Studies testing the effective placement of a device The majority of the 15 studies that evaluated whether placement of a marker caused injury or affected behavior reported one or more effects (5 out of 7 reported behavior effects, and 9 out of 13 studies reported injury and disease). Both of the studies that considered physiology reported significant effects. No study reported on effects on reproduction or growth. Two studies reported reduced survival in marked animals; three others found no effect. Radio and satellite transmitters were the most commonly studied marker (10 of 15 studies). Seven of the transmitter studies involved implanting a device and describing the implantation process and the animal’s subsequent reaction. Three other studies tested the 24  effectiveness of the device after deployment. Three studies involved passive integrated transponder (PIT) tag placement, one involved the effective placement of an acoustic tag and one involved the effectiveness of marking with fluorescent paste. Sea otters were the most commonly studied species in Category B, most likely because of their declining numbers and the need to develop a marker that does not affect the natural water-repellent pelage of the animal (Hatfield and Rathbun 1996).  2.3.2 Marking and tagging devices used Four types of marking and tagging methods dominated the marine mammal literature: 1) external marking devices (15 studies), 2) implanted tags for long-term marking (12 studies), 3) hot- and cold-iron branding (7 studies), and 4) visual tags (6 studies). In Table 2.1, the durations of time the animal wore the marking device (temporary, semi-permanent, or permanent) are reported based on classification system proposed by Mellor et al. (2004). Additionally, we classify the level of tissue manipulation or destruction involved with the placement of the device or mark as minimal (tissue manipulation but no tissue destruction), moderate (moderate tissue manipulation and or tissue destruction), or severe (severe tissue manipulation or destruction; categories based on guidelines in CCAC, 2003). Four studies assessed multiple marking devices (Irvine et al. 1982; Garshelis and Siniff 1983; Baker and Johanos 2002; Hasting et al. 2009). Studies grouped into Categories (A) and (B) showed a number of commonalities by type of marking method used.  25  2.3.2.1 External marking devices Marking devices can be secured to marine mammals through a body part (e.g., cetacean transmitter attachment using pins placed through the dorsal fin or ridge), with suction cup tags (also referred to as remora tags), by placing it around the animal’s neck or ankle, or by gluing the device to the animal’s pelage. Thirteen of the 15 studies assessing the attachment of external devices, such as radio transmitters and time-depth recorders, have focused on behavioral effects. Physiological effects (n = 2), injuries (n = 6), mortality (n = 4), and reproductive and growth rate effects (n = 2) were addressed in fewer studies. Non-lethal firing of projectiles is commonly used to attach devices onto cetaceans. Cetacean behavioral responses to the attachment of external devices has included aberrant swimming behavior when attached through the dorsal fin using bolts (Irvine et al. 1982), changes in the frequency of leaps and group speed after suction cup attachment (Schneider and Baird 1998) and flinching, tail slapping, rapid swimming and more surfacing attempts after suction cup attachment (Hanson and Baird 1998; Hooker et al. 2001; Blomqvist and Amundin 2004). External devices deployed by implantation into the skin or blubber of whales has shown minimal behavioral effects, including skin-twitches followed by shallow dives or no response (Mate and Harvey 1983; Watkins and Tyack 1991; Goodyear 1993), or breaching and rapidly accelerating upon tagging (one whale, Goodyear 1993). Some whales react to missed tagging attempts by swimming away, raising their heads or backs out of the water, defecating, and quickly submerging — most likely because of the splash the device made in the water (Watkins and Tyack 1991). One study concluded that anchors used to attach the tags did not appear to cause severe damage; one whale that lost its tag showed swelling, but no sign of laceration, around the tag entry point (Mate and Harvey 1983).  26  Most studies examining the behavioral effects of external devices on cetaceans were conducted in the wild and did not record pre-tagging behavior. One exception is a study on a single harbor porpoise (Phocoena phocoena; Geersten et al. 2004) that reported changes in log-rolling behavior, roll duration, dive duration, daily food intake and surfacing areas after a radio transmitter was attached through the dorsal fin. Epoxy glue has been used in four studies to attach external devices to the pelage of animals (Walker and Boveng 1995; Baker and Johanos 2002; McCafferty et al. 2007; McMahon et al. 2008). Due to its thermosetting components, epoxy glue has the potential to cause thermal burns and may cause reactions if in contact with the skin of an animal. Unfortunately, no study has been published addressing these effects. Studies have instead focused on broader effects, such as maternal foraging and attendance behavior (Walker and Boveng 1995) and survival and migration (Baker and Johanos 2002). For example, Antarctic fur seals (Arctocephalus gazella) fitted with both time-depth recorders and radio transmitters have increased foraging-trip and nursing-visit durations compared with animals carrying only radio transmitters (Walker and Boveng 1995). The effects of the radio transmitter alone were not identified. Another study using devices attached with epoxy glue examined the effects of research handling, including blood sampling, flipper tagging and the placement of time-depth recorders, data loggers and video recorders, on the migratory behavior, survival and body condition of Hawaiian monk seals (Monachus schauinslandi), and found no difference between control and handled animals (Baker and Johanos 2002). There was, however, no direct assessment of how the attachment of devices affected the behavior or foraging success of the animals.  27  Alternative methods of attaching devices include neck and ankle collars. These methods of attachment were found to be highly detrimental to the sea otters, causing severe constriction and one death attributable to the device (Garshelis and Siniff 1983). Flipper transmitters were believed to be the least likely to cause fatal injury, but effects on survival could not be determined. Flipper transmitters did result in injuries (broken middle digits and slits in the webbing of the otter’s flipper) and altered behavior (otters pulled at the transmitter and held their flipper out of the water), and were typically lost within 3 months (Garshelis and Siniff 1983). The authors tested the effectiveness of their device by using 144 sea otters fitted with radio transmitters placed on their rear flipper with the use of steel bolts. We suggest that future research should make an effort to use the fewest number of animals to meet research objectives (i.e., power analyses should be conducted) and the marking method should be tested on a subset of animals in the study species of interest prior to larger scale deployment. In another flipper attachment study 22 of 75 tagged otters were never seen again, and 18 of the 53 otters that were seen after tagging had sustained flipper damage because of the transmitter attachment (Hatfield and Rathbun 1996). Flippers carrying transmitters were seen drooping unnaturally at the tip, due perhaps to increased drag. Other studies assessing survival and reproduction after the placement of an external marking device have found no effect (Baker and Johanos 2002; Martin et al. 2006; McMahon et al. 2008). These results may be specific to the particular marking devices and species tested, and should not be used as justification for using similar techniques on other species. The animals used in these studies may have been particularly robust (as suggested by Martin et al. 2006). Results may also vary with environmental conditions, and these should be evaluated before deployment of full-scale marking programs.  28  Only two studies reported on the physiological effects of the attachment of external marking devices. These studies found changes in temperature distribution around the attachment site (McCafferty et al. 2007), increased heart rate at the time of device attachment, as well as a reddish exudate at the attachment site (Geersten et al. 2004).  2.3.2.2 Implanted tags One limitation of external attachment of telemetry devices on aquatic mammals is the high rate of instrument loss due to physical damage and annual molts, typically limiting the monitoring of marked animals to weeks or months. Longer-term monitoring may require implanting devices into the animal, for example, through intraperitoneal or subcutaneous placements. Three of the 12 studies looking at the effects of internal devices reported behavioral effects (Mellish et al. 2007a; Horning et al. 2008; Walker et al. 2009 - Chapter 3), three reported physiological effects (Mellish et al. 2007a; Lander et al. 2005; Green et al. 2009), and four examined survival (Williams and Siniff 1983; Ralls et al. 1989; Galimberti et al. 2000; Horning et al. 2008). Nine of the 12 studies assessed the impacts of the device on injury and disease. All of these studies fell into Category B (i.e., tested the feasibility of marking and reported incidentally on the effects on the animal). No studies have considered the effects of internal placement of marking devices on reproduction or growth. Developments in technology have allowed researchers to track animals for longer periods. For example, the Life History Transmitter (LHX tag) allows life-long data to be collected on dive behavior, pressure, motion, light levels, temperature and conductivity (Horning and Hill 2005). Three studies have reported the effects of intraperitoneal  29  implantation of LHX tags in sea lions (Mellish et al. 2007a; Horning et al. 2008; Walker et al. 2009 – Chapter 3). Horning et al. (2008) looked at the feasibility of the surgical technique used for the deployment of LHX tags in California sea lions (Zalophus californianus) and Steller sea lions. All sea lions recovered well after surgery, with minimal swelling around the incision site. Physiological effects of the implantation included increased levels of acutephase proteins (i.e., indicators of infection, inflammation or tissue trauma) at 2 wk postsurgery, with levels returning to baseline within 6 wk (Mellish et al. 2007a). The first study to address pain in a marine mammal was conducted on Steller sea lions after the LHX abdominal surgery (Walker et al. 2009 – Chapter 3). Behavioral responses in the days after abdominal surgery included changes in back arching, standing, locomotion, time alert, lying time, and time spent with pressure on the belly. Sea lion behaviors were still affected 12 d after surgery suggesting the need for more effective analgesic methods for this procedure. Dive behavior was also recorded post-release. LHX implanted individuals had similar dive depth, duration, frequency and dispersal distances when compared to free-ranging non-LHX individuals (Mellish et al. 2007a). Similar to Mate et al. (2007) which gives an account of one laboratory’s experience with the development of satellite-monitored radio tag technology for whales, the LHX studies have used a variety of approaches to understand the impacts of the device on the animals. We suggest that these provide good models for future research on the effectiveness and the effects of tagging procedures. Several studies have looked at the feasibility of placing internal devices, including subcutaneous and internal placement of radio, satellite and sonic transmitters (Garshelis and Siniff 1983; Williams and Siniff 1983; Mulcahy and Garner 1999; Lander et al. 2005; Green et al. 2009) and passive integrated transponders (PIT) tags (Thomas et al. 1987; Wright et al.  30  1998; Galimberti et al. 2000). The first attempts to implant subcutaneous radio transmitters were with sea otters, some of which died within 2 to 5 d after implantation (Garshelis and Siniff 1983; Williams and Siniff 1983). One necropsy revealed a contusion on the abdominal wall with subcutaneous hemorrhaging, and led to the recommendation that radio transmitters be placed in the intraperitoneal cavity (Williams and Siniff 1983). The authors speculated that this placement would not impede the otter physically or behaviorally, but no data were provided in support. In another study, 5 of 40 sea otters implanted intraperitoneally with radio transmitters died within 223 d of the surgery (Ralls et al. 1989), but no evidence of adhesions or intestinal obstructions were found upon necropsy. The extent to which the implanted devices contributed to these deaths could not be determined. The subcutaneous implantation of radio and satellite transmitters has been reported in polar bears (Ursus maritimus; Mulcahy and Garner 1999) and harbor seals (Phoca vitulina; Lander et al. 2005). Results showed that implantation caused exudate drainage and discharge. In harbor seals, the radio transmitters were either encapsulated in a physiologically compatible wax coating or an inert resin. Wound healing varied; animals implanted with the resin-coated transmitters were more likely to develop wound discharge and openings near the incision in comparison to animals implanted with the wax-coated transmitters. Although implantation studies in marine mammals began in the 1980s, techniques and transmitters have evolved. With this evolution, one must consider if the device to be implanted is compatible biologically with the study species. For example, a study assessing the success of implanting heart rate loggers into California sea lions (Zalophus californianus; n = 3) and northern elephant seals (Mirounga angustirostris; n =3), found very different responses in the two species (Green et al. 2009). Sea lions had little swelling and no exudate  31  after implantation, but all 3 elephant seals showed a sufficient inflammatory response that the data loggers were removed. This study demonstrates that tagging methods that are successful on one species may not work well for another. Another method used to individually identify animals is the PIT tag. This consists of an electromagnetic coil and microchip (programmable with a unique code) that emits a signal when scanned with electromagnetic energy (Nietfeld et al. 1994). The tags are placed subcutaneously using a hypodermic needle and syringe and can only be read by a receiver placed close to the individual (e.g., within 20 cm). No battery is required since the chip is activated by the electromagnetic energy from the reader. This tagging method is less invasive than surgical or intra-abdominal implantation due to the small size of the device. PIT tag placement and response has been successfully measured in sea otters (Thomas et al. 1987), manatees (Trichechus manatus; Wright et al. 1998), and southern elephant seals (Galimberti et al. 2000). No tissue reactions to tag placement were found in sea otters and southern elephant seals, nor have differences been noted in survival between PIT-tagged and non PITtagged individuals (Galimberti et al. 2000). PIT tag injection sites in manatees were slightly raised and hard, with minor scarring present (Wright et al. 1998). However, the manatees were also freeze-branded and the PIT tags were injected immediately in the center of a freeze-branded area — this combination may have produced the tissue reaction.  2.3.2.3 Hot- and cold-iron branding Cattle and horses have been branded for centuries (Macpherson and Penner 1967). Branding has been modified for use in other mammals such as seals and sea lions and nondomesticated ungulates (Merrick et al. 1996; Nietfeld et al. 1994). Branding can provide a  32  mark that remains visible throughout the animal’s life. There has been intense debate as to whether branding should be used as a marking method for marine mammals. Public concern has prompted lawsuits and the revocation and suspension of research permits for hot-iron branding of some pinniped species (Green and Bradshaw 2004; Dalton 2005). Hot-iron branding uses metal branding irons of various letters, numbers or shapes that are heated until red hot and then applied to the animal’s skin with firm even pressure for between 2 to 7 s (Erickson et al. 1993; Merrick et al. 1996; Wells 2002). Hair may be removed prior to application and the branded site wiped dry to facilitate a clear uniform brand (Erickson et al. 1993; Gentry and Holt 1982). The areas commonly branded are the upper shoulder or back on pinnipeds, and the dorsal fin on dolphins (Nietfeld et al. 1994; Wells 2002). Animals must be restrained and are sometimes branded while under general gas anesthesia. Both hot-iron and freeze branding have been studied in cattle (Lay et al. 1992a, b; Schwartzkopf-Genswein et al. 1997a, b, 1998), and a few studies have addressed the physiological effects of branding in pinnipeds (Daoust et al. 2006; McMahon et al. 2006b; Mellish et al. 2007b). In freeze branding, irons are cooled in a dry ice-alcohol solution to -79ºC or with liquid nitrogen to approximately -200ºC and are held in place on the animal for 20 to 60 s (Macpherson and Penner 1967; Nietfeld et al. 1994; Daoust et al. 2006). While hot-iron brands burn through the dermal layers and disrupt the hair follicles preventing new hair growth, freeze branding damages the pigment-producing melanocytes but leaves the hair follicles intact allowing for regenerative growth of white hair (Macpherson and Penner 1967; Nietfeld et al. 1994; Wells 2002; Daoust et al. 2006). The results produced by the different  33  brands have been shown to vary by species. There is controversy over whether freeze branding is a more humane method of branding; this is based on clear signs of less acute pain with freeze branding with cattle (Lay et al. 1992a, b; Schwartzkopf-Genswein et al. 1997a, c). Studies on harbor seals and cattle have attempted to determine which method produces higher quality brands and which is a more effective and humane method (Lay et al. 1992a, b; Schwartzkopf-Genswein et al. 1997a, b; Daoust et al. 2006). To date, six studies have examined the effects of branding in three marine mammal species. One of these studies assessed physiological responses to branding (Mellish et al. 2007b), two assessed injuries and wound healing following branding (van den Hoff et al. 2004; Daoust et al. 2006) and two assessed survival (McMahon et al. 2006b; Hastings et al. 2009). One study has looked at the behavioral effects of branding (Walker et al. 2010 – Chapter 5) and no study has addressed how branding affects growth and reproduction. Sea lions display pain-related behaviors after hot-iron branding (Walker et al. 2010 – Chapter 5). Specifically, in the 3 days after branding sea lions spend more time grooming their branded area, less time with pressure on their branded side, and less time in the pool and in locomotion. These results suggest that alternative analgesia protocols are required to help mitigate the witnessed pain responses. Branding juvenile Steller sea lions with hot-irons produces a systemic inflammatory response as evidenced by changes in peripheral blood values, with levels returning to baseline 7-8 wk post-branding (Mellish et al. 2007b). This study found no significant differences in serum cortisol levels, but the initial rise may have been missed, as cortisol was sampled only 90 min after branding. Detecting a peak in cortisol levels require repeat samples at a frequency representative of the pattern of the entire response. Comparing  34  before-treatment values with samples taken at arbitrary later times can be misleading (Mellor and Stafford 2004). Wound healing patterns seem to vary among species (van den Hoff et al. 2004; Daoust et al. 2006). Among hot-iron branded southern elephant seal pups, there was a strong positive correlation between brand wound healing, brand readability and peripheral skin damage (van den Hoff et al. 2004). Brands with more peripheral skin damage had longer healing times, but most brands were completely healed within one year, with the molting process contributing to the healing process. Field observations revealed that in harbor seal pups coldiron brands healed faster, but hot-iron brands provided a more permanently legible brand (Daoust et al. 2006). Prolonged wound healing of brands may cause animals chronic pain or other behavioral changes, however no study has addressed this issue. Effects of branding on survival have been studied in southern elephant seals (McMahon et al. 2006b) and Steller sea lion pups (Hastings et al. 2009). No difference was detected in the survival of hot- and cold-iron branded elephant seals compared with individuals that were only flipper tagged, but none of the cold-iron brands were readable within one year of branding (McMahon et al. 2006b). A study assessing the weekly survival of Steller sea lion pups in the 12 wk after branding estimated that mortality of pups that could be attributed to branding was 0.5–0.7% (Hastings et al. 2009). Overall survival for the 12 wk post-branding was estimated at 0.868 and varied little with sex, year, and capture area. Interestingly, larger female pups had higher mortality rates than smaller female pups; male pups showed the opposite pattern.  35  2.3.2.4 Visual tags A variety of “visual tags” varying in color, shape, material (plastic, aluminum, steel or other alloy) and size have been used to mark marine mammals. Some tags are self-locking and others use rivets to attach to the animal. The types of tags used depend on re-sight requirements and whether they will be placed inter-digitally on the flippers, in the axillary webbing, on the dorsal fin or through the animal’s ear. To date, three of five studies designed to evaluate visual tags have reported behavioral effects, three have reported on injury and disease, and three on survival effects. Only one study has examined growth effects and none has assessed physiological effects. Only one of the three studies assessing the behavioral responses to visual tags found an effect. In this study of weaned Hawaiian monk seals, tagged animals hauled out further from the marking site than untagged animals (Henderson and Johanos 1988). Another study showed migration rates of Hawaiian monk seals were not influenced by flipper tagging (Baker and Johanos 2002). Similarly, there was no segregation or rejection between unmarked northern fur seals and animals marked with fluorescent pelage paste (Griben 1984). The effects of visual tags on injury and disease include the destruction of tissue at the site of tag attachment (Irvine et al. 1982), as well as tearing associated with tag loss (Henderson and Johanos 1988). Tissue biopsies from northern fur seals marked with fluorescent paste showed no histological abnormalities between painted and unpainted regions of the animals (Griben 1984).  36  None of the three studies assessing survival found an effect of visual tags (Hawaiian monk seals: Henderson and Johanos 1988 and Baker and Johanos 2002; Steller sea lions: Hasting et al. 2009). Trites (1991) re-evaluated data collected from 1957 to 1966 to determine whether flipper tagging and marking by slicing off the flipper tip had a significant effect on growth rates in northern fur seal pups (Callorhinus ursinus). A previous assessment of the data by Abegglen et al. (1957) concluded that marking reduced pup growth rates. On re-evaluating these data, Trites (1991) found that tagged and untagged pups grew at the same rate. He suggested that differences in weight may have been due to inadvertently choosing pups smaller in size and hence more easily captured. This study illustrates how sampling bias can influence results.  2.4 Discussion and research recommendations This section briefly summarizes key findings and identifies needs for further research. Of the 36 studies identified in the literature that specifically addressed marking and tagging, 19 were published since 2000 (Fig. 2.1). This trend may reflect increased oversight by institutional animal care and use committees requiring that researchers assess these effects. The trend may also be due to increased public scrutiny over different marking devices, as witnessed with the hot-iron branding controversy (Green and Bradshaw 2004; Dalton 2005). Research is beginning to recognize that marking and tagging studies need to consider not only the population level effects, but also the welfare of individual animals. In the past 10 years, more research has focused on assessing the direct effects of marking and tagging  37  devices (14 of the 21 studies from Category A were conducted in 2000s, compared with 5 of the 15 studies from Category B). Awareness of marking effects has led to changes in marking procedures. For example, the U.S. National Marine Fisheries Service concluded that northern fur seals that were tagged and had their flippers notched (by removing a portion of the flipper) had a lower survival rate than animals that were tagged but did not receive the flipper notch (NMFS 1970). On this basis, flipper notching is no longer used in field research. Hot-iron branding of elephant seals at Macquarie Island and sea lions in New Zealand has been suspended due to allegations that branding reduces survival, causes animals to lose body condition, and that brands do not heal well and are not always readable (McMahon 2007). Although no study has found decreased survival after branding (e.g., in comparison with those that had only been flipper-tagged; McMahon et al. 2006b), the controversy and subsequent permit suspensions speak to the weight of public concern.  2.4.1 What does the current research show? Research on the effects of externally attached devices has shown behavioral reactions (e.g., changes in swimming behaviors) and injury from placement of the device (e.g., bolt migration, constriction and swelling at attachment site). Studies on internal devices have shown physiological responses (e.g., increased acute-phase proteins), injuries (e.g., subcutaneous hemorrhaging, wound discharge), behavioral pain responses and decreased survival. Studies on hot- and cold-iron branding have also shown physiological effects (e.g., elevated white blood cell counts, platelets and acute phase proteins) and injury (e.g., delayed wound healing and tissue damage), but no differences in survival. One study assessed the  38  behavioral responses to hot-iron branding and found that animals displayed pain-related behaviors in the days following branding. Research on visual tags has shown behavioral effects (e.g., changes in haul out behavior) and injury (e.g., tissue damage due to tag loss, skin abrasions), but no effect on survival or growth rate. With the exception of survival studies, most research on marking has focused on short-term effects.  2.4.2 Where are the gaps in the literature? There have been numerous requests for marker evaluation studies on marine mammals (see Seber 1982; Murray and Fuller 2000; McMahon et al. 2006a; Beausoleil and Mellor 2007). Assessing the effects of marking and capture techniques should be an important component of marine mammal research. Permit -granting agencies often require discussion on the implications of the research techniques employed, which requires proper analysis of marking effects. Wilson and McMahon (2006) suggest, “measures to quantify the stress of capture and device attachment in wild animals should routinely be included in proposals for field work”. It is important to recognize the effects that marking and capture devices can have on the individual animal. Major gaps exist in understanding whether marking devices impede natural behaviors such as movement and feeding patterns, growth, reproduction, and health, and whether marine mammals experience pain and distress during and after marking. To date, the studies on marker effects conducted with marine mammals have mainly focused on the immediate effects on behavior and injury. This review illustrates some inconsistency in how researchers design studies and report findings. There is no standardized method for reporting marking procedures and  39  effects in marine mammals. For example, only 20 of the 36 studies in this review reported whether the data were subjected to any kind of statistical analysis (Table 2.1). We strongly recommend that future research studies include a priori hypotheses, complete methodology, and statistical tests of the hypotheses. Pain management protocols used during the marking procedure were mentioned only in a few studies, even though 25 of the 36 studies were classified as involving moderate to severe levels of tissue manipulation and destruction. Typically wildlife research goals value the needs of the ecosystem over individual animal welfare (Farnsworth and Rosovsky 1993; Fraser 1999). However, wildlife care and use guidelines, such as those of the Canadian Council on Animal Care, recommend that researchers use analgesia and anesthetics for invasive procedures (CCAC 2003). If pain is present due to research-related injury, then researchers should work with veterinarians to reduce the animals’ suffering. Pain can also affect many aspects of an animal’s normal functioning, so reducing animal pain at the time of marking or tagging has the potential to improve the quality of the scientific data.  2.4.3 Where do we go from here? The studies reviewed above reveal different limitations associated with the different marking methods, and suggest that researchers need to assess the cost to the animal when considering marking methods. Factors to consider are the length of time the marker lasts on the animal (influenced by loss of the tag over time from being ripped out or falling off due to molting, and by living conditions of the animal), as well as transmitter malfunction and battery life. Complications from capture and handling or anesthesia, as well as the resources required for effective follow up, also need to be considered. For example, surgical  40  implantation of transmitters is an effective way to overcome transmitter loss due to molting, but require surgery for the animal and longer handling times. It is important that the marker placed on the animal does not confound data collection and interpretation. Some researchers recognize that proper analysis of marking effects is needed to be confident that data collected from the marked individual is representative of the unmarked individuals in the population (e.g., Irvine et al. 1982). Unfortunately, it is often difficult to follow unmarked individuals in the wild. Studies comparing unmarked and marked animals can be conducted in a captive setting or where natural markings are distinguishable on individual animals (e.g., sea lions individually identifiable through unique scars, fungal patches, or other distinct markings, Maniscalco et al. 2006; grey seals identified by natural pelage markings, Vincent et al. 2001; small cetaceans identified by natural markings, Wursig and Jefferson 1990). Telemetry devices are becoming more accessible to wildlife researchers. However, the body may reject marking devices (e.g., Lander et al. 2005 and Green et al. 2009) especially when they are not encased in biocompatible material. The transmitter housing may break causing battery leakage inside the animal and eventual tag failure. Most studies do not report on the biocompatibility of materials used to encase the tags. We suggest that future studies report material biocompatibility for the study species and marking device used. Tagging methods that are successful on one species may not work well on other species; for example, heart rate data loggers are rejected in elephant seals but not in sea lions (Green et al. 2009). Tags should therefore be tested on the study species before full-scale deployment in field research. The placement of a marker also needs to be considered in relation to the animal’s behavior (i.e., mating, fighting) to reduce risk of marker loss. Stages  41  of an animal’s life can also affect marking success. For example, animals instrumented during critical periods, such as lactation, may exhibit different behavioral and physiological effects and reduce biological functioning (Walker and Boveng 1995). Study designs need to consider the different life-history stages of the animal and how this can affect both the animal and the data collected. Recent research has focused on the development of tags used in cetaceans through the use of computer simulations (Pavlov et al. 2007). This technique allows for a tag to be developed that minimizes the overall impact on the animal and potentially allows researchers to obtain more reliable and higher quality data. Tags built in this fashion should still go through test periods and biocompatibility trials before large-scale deployment.  2.4.4 Guiding principles for minimizing marking impacts Existing guidelines to minimize the impact of a mark on an individual, while ensuring reliable identification, include the following six criteria: (1) the marking should cause little or no effect on the animal’s anatomy and physiology, in both the short term and the long term (e.g., the animal should not experience pain or distress, prolonged wound healing time or disease), (2) the marking should not interfere with the animal’s ability to perform its natural behavior including foraging, breeding and locomotion, (3) the marking should be readable and visible, (4) the marking should not attract predators or affect potential mates or other con-specifics, (5) the mark should persist long enough to meet the research objectives, and (6) extensive handling should be avoided when applying marks (Cook 1943; Friend et al. 1994; Nietfeld et al. 1994; Murray and Fuller 2000; van def Hoff et al. 2004).  42  Additionally, we propose: (7) the fewest number of animals should be marked to meet the research objectives (i.e., power analyses should be conducted, and reported, to determine appropriate sample sizes prior to marking), (8) a pain management plan should be developed with a veterinarian, potentially including the use of appropriate anesthesia and post-operative analgesics, (9) the marking device should be tested on a subset of animals in the study species before larger scale deployment, (10) the marked population should be compared to an unmarked control population (e.g., using natural markings on the animal’s pelage) or research should conduct observations before and after a marking procedure, whenever possible, to document marking effects, and (11) markings should only be carried out by trained individuals skilled in the marking procedure. In addition to the marking guidelines listed above, we recommend that future studies monitor and report the following key parameters when conducting marking and tagging effect studies on marine mammals: (a) methods used to assess sample size such as power analyses, (b) issues with restraint or application of the device or mark, (c) anesthetic and analgesia agents used (if none were used then provide justification), (d) why the researchers felt the tag or mark they chose was appropriate for their research objectives and whether there are alternative, less invasive methods available (i.e., were the Three Rs of research; replacement, refinement, and reduction, considered; Russell and Burch 1959), (e) complete methodology for the placement of marking device, in addition to any additional tissue or blood sampling that occurred during the handling procedure, (f) the total time the animal was handled including the placement of the device, (g) the total time spent monitoring the animal, (h) level of invasiveness of the procedure based on the degree of tissue manipulation or destruction (e.g., as recognized by CACC categories), and (i) the statistical analyses  43  conducted on the data. Most importantly, as stated by Hooker et al. (2007) we also encourage authors to publish results of both best practices and results that were less favorable (i.e., negative impacts on the animal or the data), as these types of studies often go unpublished.  2.5 Conclusions To summarize, our review shows (a) research on marker effects has primarily focused on behavioral responses, such as swimming behavior, (b) few studies have addressed the effects of markers on reproduction or growth, (c) only two studies to date have addressed the pain and distress related to marking, (d) and no studies designed to show the effects of markings on survival have demonstrated reduced life-expectancy due to marking. We recommend that researchers standardize reporting of their findings using the guidelines proposed in this review. Future research on marine mammals under controlled conditions is required to document acute effects of marking, including pain and distress, and to better understand longer-term effects on health and disease, growth, reproduction, and survival.  2.6 Acknowledgments We thank David Fraser for his insightful comments on early versions of our manuscript. Funding for K.A. Walker came from University of British Columbia (UBC), the Animal Welfare Program at UBC and by its donors listed at http://www.landfood.ubc.ca/animalwelfare.  44  2.7 References Abegglen, C.E., A.Y. Roppel and F. Wilke. 1957. Alaska fur seal investigations Pribilof Islands, Alaska: report of field activities June-September 1957. U.S. Fish and Wildlife Service, Bureau of Commercial Fisheries, Section of Marine Mammal Research Seattle, WA. 162pp. Andrews, R.D., D.G. Calkins, R.W. Davis, B.L. Norcross, K. Peijnenberg and A.W. Trites. 2002. Foraging behavior and energetics of adult female Steller sea lions. Pages 19-22 in Steller Sea Lion Decline: Is it Food II? D. DeMaster and S. Atkinson, eds. Alaska Sea Grant, AK-SG-02-02, Fairbanks. Baker, G.B., L.F. Lumsden, E.B. Dettmann, N.K. Schedvin, M. Schulz, D. Watkins and L. Jansen. 2001. The effect of forearm bands on insectivorous bats (Microchiroptera) in Australia. Wildlife Research 28: 229-237. Baker, J. D. and T.C. Johanos. 2002. Effects of research handling on the endangered Hawaiian Monk seal. Marine Mammal Science 182: 500-512. Bakken, G.S., P.S. Reynolds, K.P. Kenow, C.E. Korschgen and A.F. Boysen. 1996. Thermoregulatory effects of radiotelemetry transmitters on mallard ducklings. Journal of Wildlife Management 60: 669-678. Bannasch, R., R.P. Wilson and B. Culik. 1994. Hydrodynamic aspects of design and attachment of a back-mounted device in penguins. Journal of Experimental Biology 194: 8396. Beausoleil, N.J. and D.J. Mellor. 2007. Investigator responsibilities and animal welfare issues raised by hot branding of pinnipeds. Australian Veterinary Journal 85: 484-485. Blomqvist, C. and M. Amundin. 2004. An acoustic tag for recording directional pulsed ultrasounds aimed at free-swimming bottlenose dolphins (Tursiops truncatus) by conspecifics. Aquatic Mammals 30: 345-356. Burley, N., G. Krantzberg and P. Radman. 1982. Influence of colour-banding on the conspecific preferences of zebra finches. Animal Behaviour 30: 444-455. Canadian Council on Animal Care (CCAC). 2003. CCAC guidelines on: The care and use of wildlife. Available at http://www.ccac.ca/en/CCAC_Programs/Guidelines_Policies/GDLINES/Wildlife/Wildlife.p df. Cook, A.H. 1943. A technique for marking mammals. Journal of Mammalogy 24: 45-47.  45  Curry, B.E. 1999. Stress in mammals: the potential influence of fishery-induced stress on dolphins in the Eastern tropical pacific ocean. U.S. Department of Commerce, NOAA Technical Memorandum NMFS-SWFSC-260. Dalton, R. 2005. Animal-rights group sues over ‘disturbing’ work on sea lions. Nature 436: 315. Daoust, P.-Y., G.M. Fowler and W.T. Stobo. 2006. Comparison of the healing process in hot and cold brands applied to harbour seal pups (Phoca vitulina). Wildlife Research 33: 361372. Eagle, T.C., J. Choromanski-Norris and V.B. Kuechle. 1984. Implanting radio transmitters in mink and Franklin’s ground squirrels. Wildlife Society Bulletin 12: 180-184. Erickson, A.W., M.N. Bester and R.M. Laws. 1993. Marking techniques. Pages 89-118 in Antarctic seals: research methods and techniques. R.M. Laws, ed. Cambridge University Pres, Cambridge, UK. Farnsworth, E.J. and J. Rosovsky. 1993. The ethics of ecological field experimentation. Conservation Biology 7: 463-472. Fraser, D. 1999. Animal ethics and animal welfare science: bridging the two cultures. Applied Animal Behaviour Science 65: 171-189. Friend, M., D.E. Toweill, R.L. Brownell, V.F. Nettles, D.S. Davis and W.J. Foreyt. 1994. Guidelines for proper care and use of wildlife in field research. Pages 96-124 in Research and management techniques for wildlife and habitats. T.A. Bookhour, ed., Wildlife Society, Bethesda, MD. Galimberti, F., S. Sanvito and L. Boitani. 2000. Marking of southern elephant seals with passive integrated transponders. Marine Mammal Science 16: 500-504. Garshelis, D.L. and D.B. Siniff. 1983. Evaluation of radio-transmitter attachments for sea otters. Wildlife Society Bulletin 11: 378-383. Geersten, B.M., J. Teilmann, R.A. Kastelein, H.N.J. Vlemmix and L.A. Miller. 2004. Behaviour and physiological effects of transmitter attachments on a captive harbour porpoise (Phocoena phocoena). Journal of Cetacean Research and Management 6: 139-146. Gentry, R.L. and J.R. Holt. 1982. Equipment and techniques for handling northern fur seals. U.S. Department of Commerce, NOAA Technical Report NMFS SSRF-758. 15 pp. Goodyear, J.D. 1993. A sonic/radio tag for monitoring dive depths and underwater movements of whales. Journal of Wildlife Management 57: 503-513.  46  Green, J.J. and C.J.A. Bradshaw. 2004. The “capacity to reason” in conservation biology and policy: the southern elephant seal branding controversy. Journal for Nature Conservation 12: 25-39. Green, J.A., M. Haulena, I.L. Boyd, D. Calkins, F. Gulland, A.J. Woakes and P.J. Butler. 2009. Trial implantation of heart rate data loggers in pinnipeds. Journal of Wildlife Management 73: 115-121. Griben, M.R. 1984. A new method to mark pinnipeds as applied to the Northern fur seal. Journal of Wildlife Management 48: 945-949. Guynn, D.C., J.R. Davis and A.F. VonRecum. 1987. Pathological potential of intraperitoneal transmitter implants in beavers. Journal of Wildlife Management 51: 605-606. Hanson, M.B. and R.W. Baird. 1998. Dall’s porpoise reactions to tagging attempts using a remotely-deployed suction-cup tag. Marine Technology Society Journal 32: 18-23. Hastings, K.K., T.S. Gelatt and J.C. King. 2009. Postbranding survival of Steller sea lion pups at Lowrie Island in Southeast Alaska. Journal of Wildlife Management 73: 1040-1051. Hatfield, B.B. and G.B. Rathbun. 1996. Evaluation of a flipper-mounted transmitter on sea otters. Wildlife Society Bulletin 24: 551-554. Hedd, A., R. Gales and D. Renouf. 1995. Use of temperature telemetry to monitor ingestion by a harbour seal mother and her pup throughout lactation. Polar Biology 15: 155-160. Hemsworth, P.H. 2004. Human-livestock interaction. Pages 21-38 in The well-being of farm animals. Benson, G.J., and Rollin, B.E., eds., Blackwell Publishing, Oxford. Henderson, J.R. and T.C. Johanos. 1988. Effects of tagging on weaned Hawaiian Monk seal pups. Wildlife Society Bulletin 16: 312-317. Hernandez-Divers, S.M., G.V. Kollias, N. Abou-Madi and B.K. Hartup. 2001. Surgical technique for intra-abdominal radiotransmitter placement in North American river otters (Lontra Canadensis). Journal of Zoo and Wildlife Medicine 32: 202-205. Herráez, P., E. Sierra, M. Arbelo, J.R. Jaber, A. Espinosa de los Monteros and A. Fernández. 2007. Rhabdomyolysis and myoglobinuric nephrosis (capture myopathy) in a striped dolphin. Journal of Wildlife Diseases 43: 770-774. Hooker, S.K., R.W. Baird, S. Al-Omari, S. Gowans and H. Whitehead. 2001. Behavioral reactions of northern bottlenose whales (Hyperoodon ampullatus) to biopsy darting and tag attachment procedures. Fishery Bulletin 99: 303–308.  47  Hooker, S.K., M. Biuw, B.J. McConnell, P.J.O Miller and C.E Sparling. 2007. Bio-logging science: Logging and relaying physical and biological data using animal-attached tags. Deep Sea Research Part II 54: 177-182. Horning, M. and R.D. Hill. 2005. Designing an archival satellite transmitter for life-long deployments on oceanic vertebrates: the life history transmitter. IEEE Journal of Oceanic Engineering 30: 807-817. Horning, M., M. Haulena, P.A. Tuomi and J.E. Mellish. 2008. Intraperitoneal implantation of life-long telemetry transmitters in otariids. BMC Veterinary Research 4: 51 doi:10.1186/1746-6148-4-51. Horning, M., R. Hill, A. Woakes, M. Pendergast, L. Martin and V. Burkanov. 1999. Draft workgroup report on electronics and instrumentation engineering. In: Steller sea lion implant workshop report, D. Calkins and L. Martin, eds. Texas A&M University, P.O. Box 1675 Galveston, TX 77553, USA, 15pp. Irvine, A.B., R.S. Wells and M.D. Scott. 1982. An evaluation of techniques for tagging small odontocete cetaceans. Fishery Bulletin 80: 135-142. Lander, M.E., M. Haulena, F.M.D. Gulland and J.T. Harvey. 2005. Implantation of subcutaneous radio transmitters in the Harbor seal (Phoca vitulina). Marine Mammal Science 21: 154-161. Lay, D.C., T.H. Friend, C.L. Bowers, K.K. Grissom and O.C. Jenkins. 1992a. A comparative physiological and behavioral study of freeze and hot-iron branding using dairy cows. Journal of Animal Science 70: 1121-1125. Lay, D.C., T.H. Friend, K.K. Grissom, C.L. Bowers and M. Mal. 1992b. Effects of freeze- or hot-branding on some physiological and behavioral indicators of stress. Applied Animal Behaviour Science 33: 137-147. Macpherson, J.W. and P. Penner. 1967. Animal identification I. Liquid nitrogen branding of cattle. Canadian Journal of Comparative Medicine and Veterinary Science 31: 271-274. Maniscalco, J.M., P. Parker and S. Atkinson. 2006. Interseasonal and interannual measures of maternal care among individual Steller sea lions (Eumetopias jubatus). Journal of Mammalogy 87: 304-311. Martin, A.R., V.M.F. Da Silva and P.R. Rothery. 2006. Does radio tagging affect the survival or reproduction of small cetaceans? A test. Marine Mammal Science 22: 17-24. Mate, B.R. and J.T. Harvey. 1983. A new attachment device for radio-tagging large whales. Journal of Wildlife Management 47: 868-872.  48  Mate, B.R., R. Mesecar and B. Lagerquist. 2007. The evolution of satellite-monitored radio tags for large whales: One laboratory’s experience. Deep-Sea Research II 54: 224–247. McCafferty, D.J., J. Currie and C.E. Sparling. 2007. The effect of instrument attachment on the surface temperature of juvenile grey seals (Halichoerus grypus) as measured by infrared thermography. Deep-Sea Research II 54: 424-436. McMahon, C. 2007. Branding the sea branders: what does the research say about seal branding? Australian Veterinary Journal 85: 482-483. McMahon, C., J. van den Hoff and H. Burton. 2005. Handling intensity and the short- and long-term survival of elephant seals: addressing and quantifying research effects on wild animals. Ambio 34: 426-429. McMahon, C.R., C.J.A. Bradshaw and G.C. Hays. 2006a. Branding can be justified in vital conservation research. Nature 439: 392. McMahon, C.R., H.R. Burton, J. Vandenhoff, R. Woods and C.J. Bradshaw. 2006b. Assessing hot-iron and cryo-branding for permanently marking southern elephant seals. Journal of Wildlife Management 70: 1484-1489. McMahon, C.R., I.C. Field, C.J.A. Bradshaw, G.C. White and M.A. Hindell. 2008. Tracking and data-logging devices attached to elephant seals do not affect individual mass gain or survival. Journal of Experimental Marine Biology and Ecology 360: 71-77. Mellish, J., J. Thomton and M. Horning. 2007a. Physiological and behavioral response to intra-abdominal transmitter implantation in Steller sea lions. Journal of Experimental Marine Biology and Ecology 351: 283-293. Mellish, J., D. Hennen, J. Thomton, L. Petrauskas, S. Atkinson and D. Calkins. 2007b. Permanent marking in an endangered species: physiological response to hot branding in Steller sea lions (Eumetopias jubatus). Wildlife Research 34: 1-6. Mellor, D.J. and K.J. Stafford. 2004. Physiological and behavioural assessment of pain in ruminants: principles and caveats. Alternatives of Laboratory Animals 32: 267-271. Mellor, D.J., Beausoleil, N.J. and K.J. Stafford. 2004. Marking amphibians, reptiles and marine mammals: animal welfare, practicalities and public perceptions in New Zealand. ISBN 0-478-22563-6, Department of Conservation, Wellington, New Zealand, pp 1-55. Merrick, R. L. and T.R. Loughlin. 1997. Foraging behavior of adult female and young-of-the year Steller sea lions in Alaskan waters. Canadian Journal of Zoology 75: 776-786. Merrick, R.L., T.R. Loughlin, G.A. Antonelis and R. Hill. 1994. Use of satellite-linked telemetry to study Steller sea lion and northern fur seal foraging. Polar Research 13: 105-114.  49  Merrick, R.L., T.R. Loughlin and D.G. Calkins. 1996. Hot Branding: A technique for longterm marking of pinnipeds. U.S. Department of Commerce, NOAA Technical Memorandum NMFS-AFSC-68. Mulcahy, D.M. and G. Garner. 1999. Subcutaneous implantation of satellite transmitters with percutaneous antennae into male polar bears (Ursus maritimus). Journal of Zoo and Wildlife Medicine 30: 510-515. Murray, D.L. and M.R. Fuller. 2000. A critical review of the effects of marking on the biology of vertebrates. Pages 15-64 in Research techniques in animal ecology. L. Boitani and T.K. Fuller, eds. Columbia University Press, New York, NY. National Marine Fisheries Service, NMFS. 1970. Fur seal investigations, 1968. U.S. Dep. Commer., National Marine Fisheries Service Special Scientific Report. Fish 617. 125pp. Nietfeld, M.T., M.W. Barrett and N. Silvy. 1994. Wildlife marking techniques. Pages 140168 in Research and management techniques for wildlife and habitats, Fifth ed.; T.A. Bookhout, ed. The Wildlife Society. Paterson, J. 2007. Capture myopathy. Pages 115-121 in Zoo animal and wildlife immobilization and anesthesia. G. West, D. Heard and N. Caulkett, eds. Blackwell Publishing Ltd. Pavlov, V.V., R.P. Wilson and K. Lucke. 2007. A new approach to tag design in dolphin telemetry: Computer simulations to minimise deleterious effects. Deep-Sea Research II 54: 404-414. Pavone, L.V. and R. Boonstra. 1985. The effects of toe clipping on the survival of the meadow vole (Microtus pennsylvanicus). Canadian Journal of Zoology 63: 499-501. Pietz, P.J., G.L. Krapu, R.J. Greenwood and J.T. Lokemoen. 1993. Effects of Harness Transmitters on Behavior and Reproduction of Wild Mallards. Journal of Wildlife Management 57: 696-703. Ralls, K., D.B. Siniff, T.D. Williams and V.B. Kuechle. 1989. An intraperitoneal radio transmitter for sea otters. Marine Mammal Science 5: 376-381. Rushen, J., A.M.B. de Passillé and L. Munksgaard . 1999. Fear of people by cows and effects on milk yield, behavior, and heart rate at milking. Journal of Dairy Science 82: 720727. Russell, W.M.S and Burch R.L. 1959. The Principles of Humane Experimental Technique, 238pp. Meuthen: London, UK. Schneider, K. and R.W. Baird. 1998. Reactions of bottlenose dolphins to tagging attempts using a remotely-deployed suction-cup tag. Marine Mammal Science 14: 316-324.  50  Schwartzkopf-Genswein, K.S., J.M. Stookey and R. Welford. 1997a. Behavior of cattle during hot-iron and freeze branding and the effects on subsequent handling ease. Journal of Animal Science 75: 2064–2072. Schwartzkopf-Genswein, K.S., J.M. Stookey, E.D. Janzen and J. McKinnon. 1997b. Effects of branding on weight gain, antibiotic treatment rates and subsequent handling ease in feedlot cattle. Canadian Journal of Animal Science 77: 3661-367. Schwartzkopf-Genswein, K.S., J.M. Stookey, A.M. Depassille and J. Rushen. 1997c. Comparison of hot-iron and freeze branding on cortisol levels and pain sensitivity in beef cattle. Canadian Journal of Animal Science 77: 369-374. Schwartzkopf-Genswein, K.S., J.M. Stookey, T.G. Crowe and B.M.A. Genswein. 1998. Comparison of image analysis, exertion force, and behavior measurements for use in the assessment of beef cattle responses to hot-iron and freeze branding. Journal of Animal Science 76: 972-979. Seber, G.F. 1982. The Estimation of Animal Abundance and Related Parameters, 2nd edition. Macmillan, New York. 654 pp. Smith, T.G., B. Beck and G.A. Sleno. 1973. Capture, handling, and branding of ringed seals. Journal of Wildlife Management 37: 579-583. Spraker, T. R. 1993. Stress and capture myopathy in artiodactylids. Pages 481-488 in Zoo and Wild Animal Medicine; M. E. Fowler, ed. W. B. Saunders, Philadelphia, PA. Swenson, J.E., K. Wallin, G. Ericsson, G. Cederlund and F. Sandegren. 1999. Effects of eartagging with radiotransmitters on survival of moose calves. Journal of Wildlife Management 63: 354-358. Testa, J.W. and P. Rothery. 1992. Effectiveness of various cattle ear tags as markers for Weddell seals. Marine Mammal Science 8: 344-353. Thomas, J.A., L.H. Cornell, B.E. Joseph, T.D. Williams and S. Dreischman. 1987. An implanted transponder chip used as a tag for sea otters (Enhydra lutris). Marine Mammal Science 3: 271-274. Trites, A.W. 1991. Does tagging and handling affect the growth of northern fur seal pups (Callorhinus ursinus)? Canadian Journal of Fisheries and Aquatic Sciences 48: 2436-2442. van den Hoff, J., M.D. Sumner, I.C. Field, C.J.A. Bradshaw, H.R. Burton and C.R. McMahon. 2004. Temporal changes in the quality of hot-iron brands on elephant seal (Mirounga leonine) pups. Wildlife Research 31: 619-629.  51  Vincent, C., L. Meynier and V. Ridoux. 2001. Photo-identification in grey seals: legibility and stability of natural markings. Mammalia 65: 363-372. Walker, B.G. and P.L. Boveng. 1995. Effects of time-depth recorders on maternal foraging and attendance behavior of Antarctic fur seals (Arctocephalus gazelle). Canadian Journal of Zoology 73: 1538 -1544. Walker, K.A., M. Horning, J.E. Mellish and D.M. Weary. 2009. Behavioural responses of juvenile Steller sea lions to abdominal surgery: developing an assessment of post-operative pain. Applied Animal Behaviour Science 120: 201-207. Walker, K.A., J.E. Mellish and D.M. Weary. 2010. Behavioural responses of juvenile Steller sea lions to hot-iron branding. Applied Animal Behaviour Science 122: 58-62. Watkins, W.A. and P. Tyack. 1991. Reaction of sperm whales (Physeter catodon) to tagging with implanted sonar transponder and radio tags. Marine Mammal Science 7: 409-413. Watson, K.P. and R.A. Granger. 1998. Hydrodynamic effect of a satellite transmitter on a juvenile green turtle (Chelonia mydas). Journal of Experimental Biology 201: 2497-2505. Webster, B.A. and R.J. Brooks. 1980. Effects of radiotransmitters on the meadow vole, Microtus pennsylvanicus. Canadian Journal of Zoology 58: 997-1001. Wells, R.S. 2002. Identification Methods. Pages 601-608 in Encyclopedia of Marine Mammals; W.F. Perrin, B. Wursig and J.G.M. Thewissen, eds. Academic Press, London, UK. Williams, T.D. and D.B. Siniff. 1983. Surgical implantation of radiotelemetry devices in the sea otter. Journal of the American Veterinary Medical Association 183: 1290-1291. Wilson, R.P. and C.R. McMahon. 2006. Measuring devices on wild animals: what constitutes acceptable practice? Frontiers in Ecology and the Environment 4:147–154. Wright, I.E., S.D. Wright and J.M. Sweat. 1998. Use of passive integrated transponders (PIT) tags to identify manatees (Trichechus manatus latirostris). Marine Mammal Science 14: 641645. Wursig, B. and T.A. Jefferson. 1990. Methods of photo identification for small cetaceans. Reports of the International Whaling Commission Special Issue 12: 43-52.  52  CHAPTER 3: Behavioural responses of juvenile Steller sea lions to abdominal surgery: Developing an assessment of postoperative pain2 3.1 Introduction Considerable research has focused on painful procedures on farm animals, such as dehorning in dairy calves and castration in piglets (e.g. Weary et al., 2006); however, much less is known about pain responses in wildlife. Research on wildlife often requires the application of marking and tracking devices (Murray and Fuller, 2000) and such procedures may cause pain. For example, marine mammals are sometimes marked using hot-iron branding and followed using tracking devices that are implanted via abdominal surgery. Given the logistical difficulties of many marine mammal field studies, the opportunity to assess marking procedures has been limited or non-existent. Only recently have efforts been made to assess effects of some marking methods used (e.g. Daoust et al., 2006; Mellish et al., 2007a, b), and no study has experimentally addressed post-operative pain. In the absence of validated methods for pain assessment and treatment, some researchers may not provide, or fail to report the use of, analgesics following procedures such as hot- and coldiron branding (van den Hoff et al., 2004; Daoust et al., 2006) and surgical implantation of radio transmitters (Ralls et al., 1989). Several marine mammal species have experienced significant population declines over the past few decades, including the endangered Western population of Steller sea lions  2  A version of this chapter has been published. Walker, K.A., Horning, M., Mellish, J.E., and Weary, D.M. 2009. Behavioural responses of juvenile Steller sea lions to abdominal surgery: Developing an assessment of post-operative pain. Applied Animal Behaviour Science 120: 210-207. 53  (Eumetopias jubatus). Long-term ecological data are essential for an understanding of past and present population trajectories, which requires animals to be individually identified (e.g. hot-iron branded, flipper-tagged) and monitored over long periods of time. Despite the controversy around some of these procedures (Green and Bradshaw, 2004; Dalton, 2005), and repeated calls for studies on the effects of marking (Murray and Fuller, 2000; Beausoleil and Mellor, 2007), there has been little research on the effects of marking of marine mammals including post-operative pain.  3.1.1 Pain assessment The experience of pain is subjective and includes sensory, cognitive and affective components. As defined by the International Association for the Study of Pain (IASP), pain is ‘‘an unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage’’ (IASP, 1994). There is much interest in developing valid and reliable techniques for the objective assessment of pain in animals. Methods of pain assessment in animals include measures of general body functioning (e.g. feed intake), physiological response measures (e.g. change in cortisol levels), as well as changes in behaviour (Weary et al., 2006). Pain-related behaviours might be observed for days to weeks after a painful procedure due to tissue damage, inflammation and repair. Behavioural responses will vary among species, but may include altered posture (e.g. time spent lying down or standing), changes in specific movements (e.g. trembling, tail or earflicking, kicking, slower locomotion), reluctance to feed and lethargy. Some animals display noticeable pain-related behaviours (e.g. vocalizations by pigs during castration: White et al., 1995; kicking and abnormal lying positions in calves due to castration: Molony et al., 1995).  54  Other animals do not show such responses; stoicism may be a survival mechanism for prey species that are in danger of alerting predators. Pain-specific behaviours may occur during a procedure. For example, at the time of hot-iron branding cattle show escape-avoidance reactions (Lay et al., 1992) and behaviours such as tail flicking, kicking, and falling (Schwartzkopf-Genswein et al., 1997). Particularly relevant to the current study is research on responses following abdominal surgery. Research on rats (Roughan and Flecknell, 2001) and cats (Waran et al., 2007) have identified specific behaviours that emerge in the hours after surgery, including back arching, writhing, twitching and crouching. These behaviours were reduced or eliminated with effective doses of analgesics.  3.1.2 Aim The aim of the current study was to describe the specific behavioural responses that occur in juvenile Steller sea lions following abdominal surgery for the implantation of telemetry devices, as a first step in understanding pain responses in this marine mammal. These sea lions were captured and underwent abdominal surgery as a part of a separate larger project. However, the current permitting guidelines for this endangered species did not allow for specific treatment groups (e.g. with and without analgesics). Under these conditions, we tested the primary hypothesis that behaviour measured pre-surgery would differ from postsurgery, and these differences would return to baseline by the late post-surgery period.  55  3.2 Methods 3.2.1 Study design and animals This study was conducted at a quarantine facility at the Alaska SeaLife Center (ASLC) in Seward, AK, USA, as apart of the larger Transient Juvenile Steller Sea Lion Project (Mellish et al., 2006). The facility consists of four adjoining pools each enclosed by a metal surface haul-out area. A chain link fence surrounds each pool such that animals can be housed individually or share access to multiple pools via sliding gates, as research and husbandry protocols require. Free-ranging juvenile Steller sea lions, between 16 and 23 months of age, included in this study were captured in Prince William Sound, AK, USA, as described by Mellish et al. (2006). Animals were from two separate capture groups; Group 1 was captured in August 2007 and contained five males and Group 2 was captured in February 2008 and contained three males and one female. Animals were transported to the ASLC for up to three months of research, including abdominal surgery for implantation of Life History Transmitters (LHX) tags, and were housed together to the maximum extent possible. The sea lions were uniquely identified with symbols shaved in their fur on their dorsal side to facilitate identification prior to permanent marking. All animals were implanted with two LHX tags 5 weeks after capture. Sea lions were alternately assigned to hot-iron branding immediately following LHX tag surgery (five sea lions) or to the LHX tag surgery alone (four sea lions). All LHX implantation and hot-iron branding events were performed under the Transient Juvenile Steller Sea Lion Project (Mellish et al., 2006), with no directed or additional handling required to achieve our  56  monitoring goal for this study. All animals were released after 9 weeks in captivity. Research was approved under Institutional Animal Care and Use Committee protocols AUP07-009 (ASLC), A07-0342 (UBC), 08-26 (UAF) and NMFS permit #881-1890-01.  3.2.2 Study procedures The LHX tags used in this study are archival, satellite-linked telemetry devices specifically designed for the life long monitoring of pinnipeds and are described in detail in Horning and Hill (2005). LHX tags are cylindrical (122 mm in length, 42 mm diameter) with a mass of 115 g. The experimental protocol of the LHX tag study required two transmitters per study animal (Horning and Hill, 2005; Horning et al., 2008). LHX tags were implanted by ventral midline laparotomy into the ventrocaudal abdominal cavity under isoflurane inhalant gas anaesthesia (as described in Horning et al., 2008), with an average duration of anaesthesia 137.3 ± 6.2 min (mean ± S.E.M.). All animals received the systemic non-steroidal anti-inflammatory analgesic flunixin meglumine (Banamine®) administered at 1 mg/kg per total body mass intramuscularly into the hip region immediately prior to extubation (onset within 2 h, duration 12-24 h). No complications due to surgery were noted and all animals recovered from anaesthesia without incident. At the attending veterinarians direction, the analgesia protocol was modified midway through the study; such that four sea lions from Group 2 also received a line block local anaesthetic combination of 2 mL lidocaine and 1 mL bupivacaine (lidocaine – onset 3-5 min, duration 60-120 min; bupivacaine – onset 6-20 min, duration 200 min) via subcutaneous injection immediately prior to and alongside the first incision. This provided the unexpected  57  opportunity to compare animals with and without line block analgesia, with the researcher blind to the line block treatment. Hot-iron brand marks consisted of a combination of four numerals (each 10.2 cm high and 5.1 cm wide). Each numeral was applied to the left shoulder/flank for 2 to 4 sec each after the completion of surgery and while still under gas anaesthesia, as described by Mellish et al. (2007b).  3.2.3 Behavioural observations All behaviours were monitored for 9 days: 3 days before surgery, Days 0-2 immediately following surgery, and again in Days 10-12 after surgery (nominally pre-, postand late post-). With the exception of the day of surgery, focal sampling occurred on all animals six times a day in 10-min periods, twice during each of the following periods of the day: 09:00-11:00 h, 13:00-15:00 h and 17:00-19:00 h. On the day of surgery, focal animals were observed for 1 h after surgery (which took place within 1.5 h after being extubated from anaesthesia, while being held in a dry holding area between pools). Steller sea lions typically return to activities, such as locomotion, within 1 hr after isoflurane gas anaesthesia (Heath et al., 1997). After this first hour of observation, 10-min observations resumed. All animals were observed for the same amount of time and observations were equal across the three dayparts, with the exception of Day 10, which has missing observations from three animals. Behaviours were recorded live by one observer (KAW). This observer (KAW) had extensive experience scoring these behaviours in sea lions. The observer was sheltered from the sea lions’ view, either behind a plastic blind or via a one-way window depending upon the location of the focal animals.  58  The behaviours listed in Table 3.1, as well as lying and sitting position (dorsal, ventral or on their side) and proximity to others, were recorded using point-in-time sampling (one sample every 1 min for 10 min). Mutually exclusive behaviours included locomotion (which includes both on land and in the water), sit upright, lie down, stand, groom, and float. Mean proportion of time on ventral side was measured during the time sea lions spent sitting upright and lying down while on land. Mean proportion of time back arching was calculated from sea lions that displayed back arch behaviour on land during periods of sitting upright and lying down. Mean proportion of time spent in the pool was calculated from activities that occur in the water (i.e., locomotion, floating and foraging).  59  Table 3.1 Descriptions of behavioural activities recorded before and after LHX implant surgery.  Behaviour Description Land and water behaviours: Alert  attentive with both eyes open  Locomotion  moving on the ground or in water (i.e. swimming by actively propelling itself through the water by means of movement of the body) Land only behaviours: Sit upright  Lie down Stand Back arch  weight placed on back flippers and/or lower body, upper body lifted off the ground, front flippers touching ground but not bearing the majority of the animal’s weight sea lion is in a flat or horizontal position to the ground, while on their ventral, dorsal, or lateral side, head may be lifted or on the ground weight is distributed among all four flippers, quadrapedally, that are positioned underneath the sea lion’s body, belly lifted off the ground, head up dorsal curvature of the spine (non-linear) in the lower thoracic and lumbar vertebrae region while lying down or sitting upright, belly lifted up off the ground  Grooming: Scratch Bite  use of flipper to scrape at skin use of teeth to grip or hold an area of body, usually witnessed in a fast repetitive motion) Body rub moves part of body back and forth with friction and pressure on the ground, fence, wall, dry mat or on another sea lion Head rub moves head back and forth with friction and pressure on another area of its own body Water only behaviours: Float  suspended in the pool, either free-floating or with flippers hanging on to side of the pool  To determine sample size required for behaviours to provide information on the effects of LHX implant surgery, power calculations were computed using preliminary results from August 2007 data (using Piface Version 1.63 software). For the 11 behaviours listed in  60  Table 3.1, the analysis determined that a sample size of between three and eight individuals would be required to accurately identify the behavioural effects of LHX implant surgery. To establish if the chosen sampling method accurately represented the proportion of time spent in each of the behaviours listed in Table 3.1, a validation study was conducted on three animals for a full day before and after the procedure (during daylight hours). The estimates generated using our sampling method (six 10-min sampling periods a day) were compared to the total daily proportion of time spent in each behaviour using regression. Only behaviours with a regression coefficient of 0.80 or higher were included in the study (pooled R2 =0.94, range 0.80 – 0.99). On this basis, the behaviours sit upright, groom and float were excluded from the analysis.  3.2.4 Statistical analysis Days were calculated using 24-h periods, with Day 0 starting immediately following extubation from anaesthesia. The proportion of time spent displaying each behaviour was averaged across both the pre-surgery, post-surgery and the late post-surgery days to generate one measure per animal per period. Proportional data were outside the range of 0.3 to 0.7. Therefore, to condense the distribution and to allow for use in the statistical analyses, all data were arcsine square root transformed (Y =arcsine √p). Mixed model analysis (SAS v9.1) was conducted to test the effects of LHX implant surgery day on the various behavioural activities. The analysis included animal as a random effect and tested for linear effects of day. The model included a within-subject factor (Day: pre-, post- and late post-surgery) and two between-subjects factors (Branding: yes or no; Group: 1 or 2). The residuals from the models were tested against the basic assumptions of normality and variance homogeneity.  61  Two specified contrasts were run to compare pre- vs. post-surgery and pre- vs. late postsurgery periods. In all cases, differences were considered to be significant at P ≤ 0.05.  3.3 Results Changes in sea lion behaviour were noted for six parameters (stand, back arch, time spent on ventral side, locomotion, time spent alert, and lying time; Table 3.2). In particular, there was an effect of day for two behaviours that were rarely observed prior to surgery: standing and back arching (F2,12 = 48.18, P < 0.001 and F2,12 = 128.98, P < 0.001, respectively). Time spent standing and with the back arched was higher post-surgery than pre-surgery (F1,12 = 85.97, P < 0.001 and F1,12 = 246.92, P < 0.001, respectively; Fig. 3.1a). Standing and back arch peaked post-surgery, but still occurred in the late post-surgery period (F1,12 = 29.71, P = 0.001 and F1,12 = 68.05, P < 0.001, respectively; Fig 3.2).  62  Table 3.2 Least square means and S.E.M. for the proportion of time sea lions (n = 9) spent displaying behaviours before and after LHX implant surgery. Means and S.E.M. are the arcsine square root transformed values. Backtransformed means are provided in parentheses. Specified contrasts pre- vs. post-surgery and pre- vs. late post-surgery P-values are presented and considered significant at P ≤ 0.05.  pre-surgery post-surgery  late postsurgery  S.E.M.  pre- vs. post- pre- vs. late surgery post-surgery P-value P-value  Land and water behaviours: Alert  0.95 (0.66)  0.85 (0.56)  0.89 (0.60)  0.04  0.02  0.20  Locomotion  0.23 (0.05)  0.12 (0.01)  0.25 (0.06)  0.03  0.05  0.68  Lying down  0.71 (0.42)  0.93 (0.64)  0.96 (0.67)  0.08  0.07  0.03  on ventral side  1.56 (1.0)  0.43 (0.17)  0.46 (0.20)  0.06  < 0.001  < 0.001  Stand  0.00 (0.00)  0.26 (0.07)  0.20 (0.04)  0.03  < 0.001  0.001  Back arch  0.11 (0.01)  0.86 (0.57)  0.61 (0.33)  0.06  < 0.001  < 0.001  0.44 (0.18)  0.45 (0.19)  0.1  0.51  0.49  Water only behaviours: Time spent 0.51 (0.24) in pool  63  Figure 3.1 Least square means (± S.E.M.) for the proportion of time sea lions spent (a) displaying back arch behaviour while sitting upright and lying down while on land, (b) with pressure on the ventral side during periods of lying down or sitting upright while on land, and (c) in locomotion on both land and in the water. Means and S.E.M. are the arcsine square root transformed values. The x-axis represents time, presented as pre-surgery (average of 3 days before surgery), post-surgery (average of the 3 days immediately following surgery), and late post-surgery (average of Days 10-12 following surgery). Day effects on back arch, time on ventral side, and locomotion were significant at P < 0.001, P < 0.001 and P = 0.05, respectively.  64  (a)  Back arch  1 0.8 0.6 0.4 0.2 0  pre-surgery  (b)  post-surgery  late post-surgery  On ventral side  1.6 1.2 0.8 0.4 0  1  (c)  2  3  post-surgery  late postsurgery  Locomotion  0.4 0.3 0.2 0.1 0 pre-surgery  Days after LHX surgery 65  Group 1 Group 2  0.40  0.30  0.20  0.10  0.00 pre-surgery  post-surgery  late post-surgery  Days after LHX surgery  Figure 3.2 Least square means (± S.E.M.) for the proportion of time sea lions spent standing on land before and after LHX surgery. Means and S.E.M. are the arcsine square root transformed values. Open circles represent animals from Group 1 (n = 5) and closed squares represent animals from Group 2 (n = 4). The overall effect of day on standing behaviour was significant at P < 0.001. The interaction between line block and day on standing behaviour was significant at P = 0.047.  66  There was a significant effect of day on the time sea lions spent with pressure on their ventral side (F2,12 = 127.93, P ≤ 0.001; Fig. 3.1b). During periods of lying and sitting, sea lions spent less time with pressure on their ventral side post-surgery (F1,12 = 208.44, P ≤ 0.001); this decrease was still witnessed in the late post-surgery period (F1,12 = 172.03, P ≤ 0.001). Sea lions instead switched to lying and sitting on their left and right sides. There was an effect of day for locomotion behaviour (F 1,12 = 3.82, P = 0.05; Fig. 3.1c). When compared with the pre-surgery period, sea lions spent less time post-surgery in locomotion (F 1,12 = 4.71, P = 0.05) and this response returned to baseline by the late post-surgery period. Sea lions tended to spend less time alert and more time lying down after LHX surgery (F2,12 = 3.11, P = 0.08 and F2,12 = 2.99, P = 0.09, respectively). Specifically, sea lions spent less time alert in the post-surgery period compared with pre-surgery (F 1,12 = 7.05, P = 0.02), with this response returning to baseline by the late post-surgery period. There was a tendency for sea lions to spend more time lying down post-surgery when compared with pre-surgery (F 1,12 =  3.87, P = 0.07), with this increase from pre-surgery more evident in the late-post  surgery period (F 1,12 = 6.36, P = 0.03). There was no effect of day on time spent in the water. For all behaviours there was no effect of branding additional to that of the surgery. When comparing sea lions from Group 1 with Group 2, there was an interaction between group and day for standing behaviour (F 2,12 = 3.97, P = 0.047). Sea lions from the Group 2 spent less time standing in the post- and late post-surgery periods than the sea lions from the Group 1 (Fig. 3.2). No other group interactions were significant.  67  3.4 General discussion This study provides the first description and analysis of post-operative behavioural responses to surgery in a marine mammal. Standing, back arching, and lying time increased, and time spent resting on the ventral surface, time alert, and overall locomotion on land and water decreased in the days following abdominal surgery. Standing and back arching were never or rarely observed before surgery. Time spent with pressure on the ventral side where the incision site was located decreased following surgery; sea lions instead switched to lying and sitting on their sides. Sea lions may use these postures to reduce stimulating the area of the injury. The tissue and nerve damage, as well as local inflammation, may have increased the activity of peripheral nociceptors and peripheral or central sensitization (Vinuela-Fernandez et al., 2007). An increased sensitivity to pain, or hyperalgesia, can occur due to the local release of inflammatory mediators and cytokines and is a common feature of inflammatory pain (Coderre and Melzack, 1987). Primary hyperalgesia develops at the site of the injury while secondary hyperalgesia develops in the surrounding uninjured tissue (Meyer et al., 2006). Hyperalgesic effects in cattle with mastitis persist between 4 and 20 days depending on the severity of the mastitis (Fitzpatrick et al., 1999) and for at least 5 weeks in mice with amputated tail tips (Zhuo, 1998). In the current study, back arch and standing behaviours were both reduced, but not completely eliminated by the late post-surgery period, indicating that animals may be still recovering from surgery and hyperalgesic effects may be present. Prolonged back arch and standing responses may not only result from the surgical incision, but in response to the movements of the free-floating LHX tags within the abdominal cavity. However, rats and cats who have undergone abdominal surgery, but with  68  no internal placement of a tracking device, display similar back arching (Roughan and Flecknell, 2001, 2004) and ‘half-tucked-up’ and crouching behaviours (Waran et al., 2007). Control surgery, comparing animals that undergo surgery with LHX implants versus animals that undergo surgery but do not receive an LHX implant (i.e. incision only), would help identify the potential cause of these post-operative differences. These treatments were not possible in this study, given the current permitting restrictions (NMFS permit #881-1890-01). Pain can also affect locomotion activity (Flecknell and Liles, 1991) and lying behaviour (Hemsworth et al., 2009). In the current study we found a significant reduction in locomotion both on land and in the water in the post-surgery period, with levels returning to baseline by the late post-surgery period. Sea lions also tended to spend more time lying down in the post- and late post-surgery periods compared to the pre-surgery period. The location of the abdominal wound may play a key role in potential restriction of movements such as lifting the flippers and rotating the body. Reduced locomotion and increased lying time may also be explained by the presence of inflammation and the inflammatory pain associated with the local release of inflammatory mediators and cytokines, or may simply be due to the animals adjusting to their activity levels after being in captivity for more than a month. An incision of 9 to 12 cm in length is required to insert the LHX tag into the abdomen. Smaller incision sites may be possible for applications of smaller telemetry implants. Alternatively, a change in the location of the incision site from the ventrocaudal region to the right or left side flank may alter or reduce some of these responses. Within the limited sample size and the constraints of the behavioural comparisons performed, we found no evidence of an effect of branding additional to that of the surgery. Hot-iron branding occurred as a part of the Transient Juvenile Project as described in the  69  Methods and was not an intentional part of the design of our surgery assessment. We suggest that future studies on the effects of hot-iron branding should use a more sensitive withinsubject design and should not include animals recovering from surgery. Behavioural responses differed between Group 1 and Group 2. Sea lions in the Group 2 spent less time standing after surgery. This difference may have been due to the lidocainebupivacaine line block administered to this group, or to any effects of group composition or time of year. Well-controlled studies on the effects of different analgesic protocols are still required. The behavioural differences described above cannot be definitively associated with pain, and the study was not designed to assess how much pain the animals were experiencing. Instead, the likely association between observed behavioural changes and pain should be considered as hypotheses to be tested by specific further investigations. For example, comparing changes in time budgets before and after surgery is a useful first step, but stronger conclusions will require comparison of untreated control groups (surgery and no analgesia), and analgesia and anaesthetic control groups (with no surgery; Flecknell and Roughan, 2004). Some approaches that are ideal scientifically may not be suitable for use in an endangered species like the Steller sea lion; for example, the inclusion of untreated controls group is likely not possible from a permitting standpoint. Adjustments to analgesic protocols using the current procedures as a positive control, is a preferred option. For example, some research suggests that combined pre- and post-operative analgesic treatment is more effective than pre-operative treatment alone (Dobromylskyj et al., 2001; Waran et al., 2007). The aim in pre-emptive analgesia administration is to reduce the firing of nociceptors and thus the hyper-analgesia induced by over-sensitization caused by damaged tissues  70  (Dobromylskyj et al., 2001). Increased dosage, longevity of regional and systemic analgesics, or pre-operative analgesia may allow a reduction in post-operative pain as inferred from the observed changes in behaviour. Further research with alternative drugs and dosages is needed to accurately define a maximally effective and safe analgesia treatment protocol for this species.  3.5 Conclusion In the days after abdominal surgery, Steller sea lions spent more time with their back arched and standing, and spent less time lying on the ventral side and in locomotion. These behavioural responses suggest that the animals may be attempting to minimize post-operative pain by avoiding stimulation of the incision site. Moreover, these results suggest that these responses should be useful in monitoring pain following similar surgeries in marine mammals. Behavioural responses to surgeries suggest that additional pain management strategies (i.e. alternative analgesia, increased dosage, or pre-operative administration of analgesia) should be investigated.  3.6 Acknowledgments We are grateful to the husbandry and veterinary departments at the Alaska SeaLife Center for their support. We are also grateful to our colleagues in the Animal Welfare Program at the University of British Columbia for many discussions on pain and welfare in marine mammals. This study was supported by the Pollock Conservation Cooperative Research Center, the Animal Welfare Program at the University of British Columbia and by the Alaska SeaLife Center. 71  3.7 References Beausoleil, N.J., Mellor, D.J., 2007. Investigator responsibilities and animal welfare issues raised by hot branding of pinnipeds. Aust. Vet. J. 85, 484-485. Coderre, T.J., Melzack, R., 1987. Cutaneous hyperalgesia: contributions of the peripheral and central nervous systems to the increase in pain sensitivity after injury. Brain Res. 404, 95– 106. Dalton, R., 2005. Animal-rights group sues over ‘disturbing’ work on sea lions. Nature 436, 315. Daoust, P., Fowler, G.M., Stobo, W.T., 2006. Comparison of the healing process in hot and cold brands applied to harbour seal pups (Phoca vitulina). Wildl. Res. 33, 361-372. Dobromylskyj, P., Flecknell, P.A., Lascelles, B.D., Pascoe, P.J., Taylor, P., WatermanPearson, A., 2001. Management of postoperative and other acute pain, in: Flecknell, P., Waterman-Pearson, A. (Eds.), Pain management in animals, Harcourt Publishers Limited, London, pp. 81-145. Fitzpatrick, J.L., Young, F.J., Eckersall, P.D., Logue, D.N., Knight, C.H., Nolan, A.M., 1999. Mastitis: a painful problem. Cattle Pract. 7, 225–226. Flecknell, P. A., Liles, J.H., 1991. The effects of surgical procedures, halothane anaesthesia and nalbuphine on the locomotor activity and food and water consumption in rats. Lab. Anim. 25, 50-60. Flecknell, P.A., Roughan, J.V., 2004. Assessing pain – putting research into practice. Anim. Welf. I3, S71-75. Green, J.J., Bradshaw, C.J.A., 2004. The “capacity to reason” in conservation biology and policy: the southern elephant seal branding controversy. J. Nat. Conserv. 12, 25-39. Heath, R.B., DeLong, R., Jameson, V., Bradley, D., Spraker, T., 1997. Isoflurane anesthesia in free-ranging sea lion pups. J. Wildl. Dis. 33, 206-210. Hemsworth, P.H., Barnett, J.L., Karlen, G.M., Fisher, A.D. Butler, K.L., Arnold, N.A., 2009. Effects of mulesing and alternative procedures to mulesing on the behaviour and physiology of lambs. Appl. Anim. Behav. Sci. 117, 20-27. Horning, M., Hill, R.D., 2005. Designing an archival satellite transmitter for life-long deployments on oceanic vertebrates: the life history transmitter. IEEE J. Oceanic Engineering 30, 807-817. Horning, M., Haulena, M., Tuomi, P.A., Mellish, J.E., 2008. Intraperitoneal implantation of life-long telemetry transmitters in otariids. BMC Veterinary Research 2008, 4:51. 72  International Association for the Study of Pain (IASP), 1994. Task Force on Taxonomy, in: Merskey, H., Bogduk, N. (Eds), Classification of Chronic Pain, 2nd Edition, International Association for the Study of Pain, Seattle, USA, pp 209-214. Lay, D.C., Friend, T.H., Bowers, C.L., Grissom, K.K., Jenkins, O.C., 1992. A comparative physiological and behavioural study of freeze and hot-iron branding using dairy cows. J. Anim. Sci. 70, 1121-1125. Mellish, J., Calkins, D., Christen, D., Horning, M., Rea, L., Atkinson, S., 2006. Temporary captivity as a research tool: comprehensive study of wild pinnipeds under controlled conditions. Aquatic Mammals 32, 58-65. Mellish, J., Thomton, J., Horning, M., 2007a. Physiological and behavioural response to intra-abdominal transmitter implantation in Steller sea lions. J. Exp. Mar. Bio. Ecol. 351, 283-293. Mellish, J., Hennen, D., Thomton, J., Petrauskas, L., Atkinson, S., Calkins, D., 2007b. Permanent marking in an endangered species: physiological response to hot branding in Steller sea lions (Eumetopias jubatus). Wildl. Res. 34, 1-6. Meyer, R.A., Ringkamp, M., Campbell, J.N., Raja, S.N., 2006. Peripheral mechanisms of cutaneous nociception, in: McMahon, S.B., Koltzenburg, M. (Eds.), Wall and Melzack’s Textbook of Pain: 5th edition, Elsevier Limited, pp. 3-34. Molony, V., Kent, J.E., Robertson, I.S., 1995. Assessment of acute and chronic pain after different methods of castration of calves. Appl. Anim. Behav. Sci. 46, 33-48. Murray, D.L.,Fuller, M.R., 2000. A critical review of the effects of marking on the biology of vertebrates, in: Boitani, L., Fuller, T. (Eds.), Research techniques in animal ecology, Columbia University Press, pp. 15-64. Ralls, K., Siniff, D.B., Williams, T.D., Kuechle, V.B., 1989. An intraperitoneal radio transmitter for sea otters. Mar. Mam. Sci. 5, 376-381. Roughan, J.V., Flecknell, P.A., 2001. Behavioural effects of laparotomy and analgesic effects of ketoprofen and carprofen in rats. Pain 90, 65-74. Roughan, J.V., Flecknell, P.A., 2004. Behaviour-based assessment of the duration of laparotomy- induced abdominal pain and the analgesic effects of carprofen and buprenorphine in rats. Beh. Pharmacol. 15, 461-471. Schwartzkopf-Genswein, K.S., Stookey, J.M., Welford, R., 1997. Behaviour of cattle during hot-iron and freeze branding and the effects on subsequent handling ease. J. Anim. Sci. 75, 2064–2072.  73  van den Hoff, J., Sumner, M.D., Field, I.C., Bradshaw, C.J.A., Burton, H.R., McMahon, C.R., 2004. Temporal changes in the quality of hot-iron brands on elephant seal (Mirounga leonine) pups. Wildl. Res. 31, 619-629. Vinuela-Fernandez, I., Jones, E., Welsh, E.M, Fleetwood-Walker, S.M., 2007. Pain mechanisms and their implication for the management of pain in farm and companion animals. Vet. J. 174, 227–239. Waran, N., Best, L., Williams, V., Salinsky, J., Dale, A., Clarke, N., 2007. A preliminary study of behaviour-based indicators of pain in cats. Anim. Welf. 16(S), 105-108. Weary, D.M., Neil, L., Flower, F.C., Fraser, D., 2006. Identifying and preventing pain in animals. Appl. Anim. Behav. Sci. 100, 64-76. White, R.G., DeShazer, J.A., Tressler, C.J., Borcher, G.M., Davey, S., Waninge, A., Parkhurst, A.M., Milanuk, M.J., Clemens, E.T., 1995. Vocalization and physiological response of pigs during castration with or without a local anaesthetic. J. Anim. Sci. 73, 381386. Zhuo, M., 1998. NMDA receptor-dependent long term hyperalgesia after tail amputation in mice. Eur. J.Pharmacol. 349, 211-220.  74  CHAPTER 4: The effects of two analgesic regimes on behaviour after abdominal surgery in Steller sea lions3 4.1 Introduction Tracking animals with telemetry devices is routine in conservation research to acquire life history data (e.g., mortality, behaviour, home range use and resource selection; Hooker et al., 2007; Cooke, 2008). Tracking devices are affixed on animals using different methods, including surgical implantation in the intraperitoneal cavity (Horning et al., 2008). Guidelines for the use of animals in wildlife research require that protocols minimize pain and distress associated with the placement of the tracking device (see Friend et al., 1994; Murray and Fuller, 2000; Wilson and McMahon, 2006), but no research to date has assessed the efficacy of analgesics for treating post-operative pain in any marine mammal. To date, three studies have reported behavioural and physiological responses following abdominal surgery in Steller sea lions (Eumetopias jubatus). Horning et al. (2008) reported low morbidity and zero mortality after intra-abdominal surgery for the deployment of telemetry devices in sea lions. Physiological responses are consistent with normal wound healing processes and include a temporary acute-phase protein response with levels returning to baseline by 6 weeks after surgery (Mellish et al., 2007). Behavioural responses indicative of post-operative pain have been recorded in the days after surgery (Walker et al., 2009). The behaviours are consistent with the animals attempting to minimize stimulation of the wounded area, and suggest that the post-operative pain may not be controlled successfully by the standard treatment protocol of a single dose of the non-steroidal anti-inflammatory drug 3  A version of this chapter has been submitted for publication. Walker, K.A., Horning, M., Mellish, J.E., and Weary, D.M. 2010. The effects of two analgesic regimes on behaviour following abdominal surgery in Steller sea lions. 75  (NSAID) flunixin meglumine delivered intramuscularly. NSAIDs, potent inhibitors of cyclo-oxygenases, exhibit anti-inflammatory, analgesic and antipyretic properties. NSAIDs have been shown to reduce specific pain-related behaviours in rats and cats after abdominal surgery (back arching and writhing: Roughan and Flecknell, 2001, 2004; ‘half-tucked-up’ and crouching: Waran et al., 2007), and are thought to be useful in reducing pain after surgery in other species. However, Walker et al. (2009) found persistent behavioural changes after surgery despite treatment with the NSAID flunixin meglumine on the day of surgery. Moreover, behaviours remained different from baseline in the 3 days after surgery. These results suggest that new analgesic drugs and treatment regimes should be evaluated, including extending the duration of treatment to cover the days after surgery in which behavioural changes persist. The aim of the current study was to compare efficacy of the current standard practice for this surgery (a single dose of flunixin meglumine administered immediately after surgery, per Horning et al., 2008) with an alternative analgesic (carprofen administrated in a single dose immediately after surgery and daily thereafter for 3 days). We predicted: (1) that all animals would show behavioural changes after surgery, (2) that on Days 1-3 after surgery animals receiving the extended carprofen treatment would exhibit less pronounced behavioural changes from baseline than animals receiving a single dose of flunixin meglumine, and (3) that on Days 4-6 behavioural differences between the two groups would diminish due to cessation of analgesia.  76  4.2 Methods 4.2.1 Study design and animals This study took place at the Alaska SeaLife Center in Seward, AK, USA. Twelve juvenile sea lions (three females and nine males) aged 16 to 24 months were captured from Prince William Sound, AK, USA in August 2008 (n = 6) and May 2009 (n = 6). These individuals were housed in a specialized quarantine facility for eleven weeks for research purposes under the Transient Juvenile Steller Sea Lion Project (Mellish et al., 2006). The facility included four adjoining pools (1 x 4 m diameter and 3 x 5 m diameter, 1.5 m deep) that containing unfiltered seawater from the adjacent Resurrection Bay and separated by sliding gates. Each pool was enclosed by 122 m2 of metal surface haul-out area. Animals were housed together to the maximum extent possible. Within one week of capture, sea lions were hot-iron branded on the left side under gas anaesthesia, as described by Mellish et al. (2006), with an additional unique mark shaved into the fur on the right side to allow for individual identification. Hot-iron branding affects behaviour in the days after the procedure (Walker et al., 2010); however, most effects return to pre-branding levels within 72 h of branding. Approximately nine weeks after capture, each animal was surgically implanted with two Life-History Transmitter (LHX) tags as described by Horning et al. (2008). The LHX tags used were archival, satellite-linked telemetry devices specifically designed for life-long monitoring of pinnipeds (see Horning and Hill, 2005, for tag details). Both the LHX implantation and hot-iron branding were performed for the purposes of the Transient Juvenile Steller Sea Lion Project (Mellish et al., 2006); no additional disturbance of the animals was required for the current study. This research was approved 77  under Institutional Animal Care and Use Committee protocols AUP07-009 (ASLC), A070342 (UBC), 08-26 (UAF) and NMFS permit #881-1890-01.  4.2.2 Study treatments The LHX tags were implanted into the ventrocaudal abdominal cavity by a ventral midline laparotomy while the animal was under isoflurane inhalant gas anaesthesia (as described in Horning et al., 2008). The average incision length was 9 cm. For the induction of anaesthesia, animals were masked with 5% isoflurane in 100% oxygen, after which they were intubated. Anaesthesia was maintained with 1% to 3% isoflurane in 100% oxygen delivered via a semi-closed, partial re-breathing circuit. The average duration of anaesthesia was 140.6 ± 7.1 min (mean ± S.E.M.). All animals received a 6 ml line block local anaesthetic combination consisting of 4 ml bupivacaine (Marcaine, AstraZeneca) and 2 ml lidocaine (2% Lidocaine HCl, Hospira Inc.) (bupivacaine – onset 10-15 min, duration 3-4 h; lidocaine – onset 5-10 min, duration 1-1.5 h) via subcutaneous injection before the first incision. Sea lions were randomly assigned to one of two post-operative analgesic treatments: flunixin group (n = 6) or carprofen group (n = 5). A twelfth individual was assigned to the carprofen group but was removed from the analysis due to a reluctance to feed after the surgery. The flunxin group animals received the standard treatment used in the project; this consisted of flunixin meglumine (Banamine, Intervet) administered intramuscularly (IM) into the right shoulder region immediately before extubation (1.0 mg/kg; onset within 2 h, duration 12-24 h). Carprofen group animals received carprofen (Rimadyl, Pfizer) administered IM into the right shoulder region immediately before extubation (4.4 mg/kg;  78  onset within 2 h, duration 12-24 h), and oral carprofen (Rimadyl, Pfizer) directly placed in forage fish (4.4 mg/kg; onset within 2 h, duration 12-24 h) at 24 h, 48 h and 72 h after surgery.  4.2.3 Behavioural observations Two observers (KAW and CM), blind to analgesic treatment, monitored behaviour by live observations for 3 days before surgery and 7 days after surgery (total 10 days). Days were calculated as 24-h periods, with Day 0 starting immediately after extubation from anaesthesia. Observations occurred on all animals six times a day in 10-min periods, twice during each day part (9:00-11:00 h, 13:00-15:00 h and 17:00-19:00 h). All animals were observed for the same amount of time and observations were equal across the 3 day parts, with the exception of Day 1 (missing observations from one animal) and Day 6 (missing observations from 2 animals). On the day of surgery, animals were held for up to 2.5 h, in either a recovery box or in area with no pool access, to allow for recovery from anaesthesia (based on Heath et al., 1997, where recovery occurred within 1.5 h after extubation). After this 2.5 h post-surgical period behavioural observations resumed. Based on a previous study conducted by Walker et al. (2009), we selected behaviours related to post-operative pain (Table 4.1). Seven behaviours were recorded using instantaneous sampling (one scan every min for 10 min; Martin and Bateson, 2007). Mutually exclusive behaviours included locomotion (on land and in the water), standing, and lying. Non-mutually exclusive behaviours included in the pool, back arch, on ventral side, and alert. The occurrence of the animal on its ventral side and with its back arched were measured when sea lions were sitting upright or lying down. Time spent in the pool included  79  all activities that occurred in the water (i.e., locomotion, floating and foraging). Observers were hidden from the sea lions’ view, either behind a plastic blind or via mirror-film glass. Inter-reliability scores were estimated for pre- and post-surgery observations. For presurgery, the two observers showed a 96% agreement during the 69 observations conducted on five animals. For post-surgery, the two observers showed a 94% agreement for the 120 observations conducted on six animals.  80  Table 4.1 Descriptions of instantaneous behaviours recorded before and after abdominal surgery.  Behaviours Alert Locomotion Lying On ventral side Stand Back arch In pool  Description sea lion on land, attentive, with both eyes open moving on the ground or in water (e.g. swimming by actively propelling itself through the water by means of movement of the body) body flat on the ground; head may be lifted or on the ground lying or sitting on land with the most of their body weight and pressure on the ventral side of the body weight distributed among all four flippers; flippers positioned beneath the body; belly lifted above the ground; head raised dorsal curvature of the spine (non-linear) in the lower thoracic and lumbar region while lying or sitting upright; belly lifted off the ground submerged (more than sixty percent of the body) in the water  4.2.4 Statistical analysis The proportion of time spent engaged in each behaviour was averaged for each of 4 periods: (1) the 3 days before surgery (Pre-surgery), (2) the day of surgery when IM injections of flunixin and carprofen were administered (Day 0), (3) the 3 days post-surgery when oral carprofen was administered (Days 1-3), and (4) the 3 days after carprofen treatment was ended (Days 4-6). Proportional data were outside the range of 0.3–0.7; to condense the distribution for statistical analyses all behavioural data were transformed by the arcsine square root transformation (Y = arcsine √p). Data were analysed using a mixed model (SAS v9.1), with a compound symmetry covariance structure, with subjects (animals) specified as a random effect. The model included a repeated measure (period: Pre-surgery, Day 0, Days 1-3, Days 4-6), two between81  subjects factors (analgesic: flunxin versus carprofen; and group: August versus May), and the interaction between analgesic and period. Residuals from the model were tested against the basic assumptions of normality and variance homogeneity, and plotted against the predicted values for the model. Five specified contrasts were conducted. Three contrasts compared the Pre-surgery baseline with values on Day 0, Days 1-3, and Days 4-6. Two other contrasts evaluated the interaction between analgesic and the difference between the Pre-surgery values and those on Day 0 and on Days 1-3. Differences were considered significant at P ≤ 0.05.  4.3 Results All seven behaviours changed after abdominal surgery (Fig. 4.1), but the analgesic treatment affected only time spent lying.  82  C arprofen  F lunixin 1  (a) Alert  0.75  0.5  0.25  0 0.5  Proportion of time  0.6  1.5  2.5  3.5  4.5  2.5  3.5  4.5  2.5  3.5  (b) Locomotion  0.4  0.2  0 0.5  0.9  1.5  (c) Lying  0.6  0.3  0  0.5  1.5  P re-s urgey  Day 0  Days 1-3  P eriod 83  4.5  Days 4-6  F lunixin 1  C arprofen  (d) On ventral side  0.75  0.5  Proportion of time  0.25  0 0.5  0.4  1.5  2.5  3.5  4.5  (e) Stand  0.3  0.2  0.1  0 0.5 P re-s urgey 1.5  Day 0 2.5 Days 1-33.5 Days 4-64.5  P eriod  84  F lunixin  C arprofen  (f) Back arch  0.75  0.5  Proportion of time  0.25  0 0.5  1  1.5  2.5  3.5  4.5  (g) Time in pool  0.75  0.5  0.25  0  0.5P re-s urgey 1.5 Day 0 2.5 Days 1-33.5 Days 4-64.5  P eriod  Figure 4.1 Least square means and the S.E.M. for the proportion of time sea lions spent (a) alert, (b) locomotion, (c) lying down, (d) on ventral side, (e) stand, (f) back arch and (g) time in pool, before and after abdominal surgery. Means and S.E.M. are the arcsine square root transformed values. The x-axis represents time, presented as pre-surgery (average of 3 days before surgery), Day 0 (1st 24-h period after surgery), Day 1-3 (average of Days 1, 2, and 3 after surgery), and Days 4-6 (average of Days 4, 5, and 6 after surgery). Open circles represent animals administered carprofen (n = 5) and closed squares represent animals administered flunixin meglumine (n = 6). 85  There was an overall effect of period on the time sea lions spent alert (F3,27 = 2.94, P = 0.051) and in locomotion (F3,27 = 9.09, P < 0.001). There was no difference between the Pre-surgery and Day 0 or Days 1-3, but the sea lions spent less time alert in Days 4-6 than during the Pre-surgery observations (F1,27 = 4.74, P = 0.038). Sea lions from both treatment groups spent less time in locomotion on Day 0 (F1,27 = 13.67, P = 0.001) and on Days 4-6 (F1,27 = 22.80, P< 0.001) compared with Pre-Surgery, and tended to spend less time in locomotion on Days 1-3 (F1,27 = 3.25, P< 0.082). There was an effect of period on time sea lions spent lying down after surgery (F3,27 = 3.70, P = 0.024), with both analgesic groups spending more time lying down on Day 0 compared with Pre-surgery (F1,27 = 9.82, P = 0.004). There was a mild interaction between analgesic and the difference between the Pre-surgery and Days 1-3 (F1,27 = 3.54, P = 0.071), with flunxin animals tending to spend more time lying down during Days 1-3 than during the Pre-surgery period, and carprofen animals showing the reverse. Time spent lying down returned to Pre-surgery levels for both analgesic groups by Days 4-6. Sea lions spent less time lying or sitting with pressure on the ventral side after surgery (F3,27 = 14.28, P < 0.001). When compared with Pre-Surgery, animals spent less time with pressure on their ventral sides on Day 0 (F1,27 = 31.37, P < 0.001), Days 1-3 (F1,27 = 23.85, P < 0.001) and Days 4-6 (F1,27 = 28.13, P < 0.001). Sea lions were rarely observed standing Pre-surgery, but this behaviour became more common in the days following surgery (F3,27 = 8.02, P < 0.001). Standing behaviour differed from Pre-surgery levels on Day 0 (F1,27 = 12.07, P = 0.002), Days 1-3 (F1,27 = 18.42, P < 0.001), and Days 4-6 (F1,27 = 14.01, P = 0.001).  86  Back arch was rarely observed before abdominal surgery, but increased in both treatment groups following surgery (F3,27 = 25.98, P < 0.001). Back arch was increased from Pre-surgery levels on Day 0 (F1,27 = 52.87, P < 0.001) and on Days 1-3 (F1,27 = 52.74, P < 0.001) and was still above Pre-surgery levels on Days 4-6 (F1,27 = 49.66, P < 0.001). There was an overall effect of period on time sea lions spent in the pool (F3,27 = 3.80, P = 0.021). Both flunixin and carprofen groups spent less time in the pool in Day 0 when compared with Pre-surgery levels (F1,27 = 4.03, P = 0.054); this behaviour returned to Presurgery levels by Days 1-3.  4.4 Discussion Six of the seven behaviours recorded differed between Pre-surgery and Day 0, regardless of analgesic treatment. Five of the seven behaviours were still different from Presurgery values on Days 4-6 after surgery. Our comparison of the two treatment groups indicates that the initial post-operative IM injection of carprofen, coupled with the oral doses on Days 1-3, was no more effective than a single IM injection of flunixin meglumine in controlling these behavioural responses. The change in behaviour from Pre-surgery levels was affected by treatment for only one measure (lying down), and this difference only approached statistical significance. A single dose of carprofen has been shown to be effective for approximately 8 h in dogs and 24 h in sheep, but the pharmacokinetics of carprofen have not been studied in sea lions and variation in plasma half-life and protein binding sites makes it difficult to extrapolate dosing schedules across species (Nolan, 2000). It is possible that the delivery of  87  carprofen in the fish may have slowed absorption (Ray et al., 1979; Nolan, 2000) and perhaps weakened the effectiveness of the drug. Six of the seven behaviours differed after surgery regardless of analgesic treatment, suggesting that neither analgesic protocol was entirely effective at controlling post-operative pain. Walker et al. (2009) showed that four of these behaviours (time spent lying down, on ventral side, in back arch and standing) differed from baseline levels even 12 d after surgery. These combined results indicate that the animals exhibit altered behaviour and may experience pain for several days after abdominal surgery, and suggest the need for improved pain management protocols. Observed responses persisted through Days 4-6, but there was no dramatic shift into this period after cessation of analgesia for both treatment regimes. Increased duration and frequency of post-operative NSAID administration or the administration of a different class of NSAID may be effective, but NSAIDs alone may also be unable to control pain after abdominal surgery. Abdominal surgery involves multiple levels of tissue manipulation and is associated with considerable post-operative pain in humans (Perkins and Kehlet, 2000). Opioids are typically used to treat moderate to severe pain (Nolan, 2000), and in some surgical situations a combination of opioids and NSAIDs has been shown to be more effective than the use of NSAIDs or opioids alone (e.g., ovariohysterectomy surgery in dogs: Slingsby and Waterman-Pearson, 2001). Opioids act centrally by limiting the amount of nociceptive input to the central nervous system, while NSAIDs act peripherally to decrease inflammation thus lowering the amount of nociceptive input to the central nervous system. By simultaneously acting on different parts of the pain pathway analgesic effects should be maximized, and the amount of each analgesic required will be reduced (Dobromylskyj et al.,  88  2000). Pre-emptive analgesia may help further reduce the post-operative pain responses by reducing sensitization caused by damaged tissues. Combined pre- and post-operative analgesic treatment is more effective than pre- or post-operative treatment alone (Dobromylskyj et al., 2000; Waran et al., 2007) and merits investigation for abdominal surgery in sea lions. Future research should consider the use of such multimodal analgesic therapies, including administration of pre-, intra-, and post-operative analgesia, which could result in a greater analgesic effect and fewer adverse effects (Dobromylskyj et al., 2000; Vinuela-Fernandez et al., 2007).  4.5 Conclusions These results suggest that extended treatment with carprofen provides little or no additional pain relief above the single administration of flunixin meglumine. Future studies should examine peri-operative multimodal approaches to pain management including the use of alternate drug classes such as opioids in combination with increasing the duration and frequency of post-operative NSAID administration.  4.6 Conflict of interest None of the authors of this paper has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper.  89  4.7 Acknowledgments We thank Carly Miller for her help with data collection and Drs. Pam Tuomi and Martin Haulena for their expert advice. We are grateful to the husbandry and veterinary departments at the Alaska SeaLife Center for their support. We are also grateful to our colleagues in the Animal Welfare Program at the University of British Columbia for many discussions on pain and welfare in marine mammals. This study was supported by the Alaska SeaLife Center and by the many other donors to UBC’s Animal Welfare Program listed on our web site at http://www.landfood.ubc.ca/animalwelfare.  90  4.8 References Cooke, S.J. 2008. Biotelemetry and biologging in endangered species research and animal conservation: relevance to regional, national, and IUCN Red List threat assessments. Endangered Species Research 4, 165-185. Dobromylskyj, P., Flecknell, P.A., Lascelles, B.D., Pascoe, P.J., Taylor, P., WatermanPearson, A., 2000. Management of postoperative and other acute pain. In: Flecknell, P., Waterman-Pearson, A. (Eds.), Pain management in animals, Harcourt Publishers Limited, London, pp. 81-145. Friend, M., Toweill, D.E., Brownell, R.L., Nettles, V.F., Davis, D.S., Foreyt, W.J. 1994. Guidelines for proper care and use of wildlife in field research. In: Bookhour, T.A. (Ed.), Research and management techniques for wildlife and habitats, Wildlife Society, Bethesda, MD, pp. 96-124. Heath, R.B., DeLong, R., Jameson, V., Bradley, D., Spraker, T., 1997. Isoflurane anesthesia in free-ranging sea lion pups. Journal of Zoo and Wildife Medicine 33, 206-210. Hooker, S.K., Biuw, M., McConnell, B.J., Miller, P.J.O., Sparling, C.E. 2007. Bio-logging science: Logging and relaying physical and biological data using animal-attached tags. Deep Sea Research Part II 54, 177-182. Horning, M., Hill, R.D., 2005. Designing an archival satellite transmitter for life-long deployments on oceanic vertebrates: the life history transmitter. IEEE Journal of Oceanic Engineering 30, 807-817. Horning, M., Haulena, M., Tuomi, P.A., Mellish, J.E., 2008. Intraperitoneal implantation of life-long telemetry transmitters in otariids. BMC Veterinary Research 4, 51. http://www.biomedcentral.com/1746-6148/4/51 Martin, P., Bateson, P. 2007. Measuring behaviour: an introductory guide. Cambridge University Press, 187 pp. Mellish, J., Calkins, D., Christen, D., Horning, M., Rea, L., Atkinson, S., 2006. Temporary captivity as a research tool: comprehensive study of wild pinnipeds under controlled conditions. Aquatic Mammals 32, 58-65. Mellish, J., Thomton, J., Horning, M., 2007. Physiological and behavioral response to intraabdominal transmitter implantation in Steller sea lions. Journal of Experimental Marine Biology and Ecology 351, 283-293. Murray, D.L., Fuller, M.R., 2000. A critical review of the effects of marking on the biology of vertebrates. In: Boitani, L., Fuller, T. (Eds.), Research techniques in animal ecology, Columbia University Press, pp. 15-64.  91  Nolan, A.M., 2000. Pharmacology of analgesic drugs. In: Flecknell, P., Waterman-Pearson, A. (Eds.), Pain management in animals, Harcourt Publishers Limited, London, pp. 21-52. Perkins, F.M., Kehlet, H., 2000. Chronic pain as an outcome of surgery. Anesthesiology 93, 1123-1133. Ray, J.E., Wade, D.N., Graham, G.G., Day, R.O., 1979. Pharmacokinetics of carprofen in plasma and synovial fluid. Journal of Clinical Pharmacology 19, 635-643. Roughan, J.V., Flecknell, P.A., 2001. Behavioural effects of laparotomy and analgesic effects of ketoprofen and carprofen in rats. Pain 90, 65-74. Roughan, J.V., Flecknell, P.A., 2004. Behaviour-based assessment of the duration of laparotomy- induced abdominal pain and the analgesic effects of carprofen and buprenorphine in rats. Behavioural Pharmacology 15, 461-471. Slingsby, L.S., Waterman-Pearson, A.E., 2001. Analgesic effects in dogs of carprofen and pethidine together compared with the effects of either drug alone. Veterinary Record 148, 441-444. Vinuela-Fernandez, I., Jones, E., Welsh, E.M, Fleetwood-Walker, S.M., 2007. Pain mechanisms and their implication for the management of pain in farm and companion animals. The Veterinary Journal 174, 227–239. Walker, K.A., Mellish, J.E.,Weary, D.M., 2010.Behavioural responses of juvenile Steller sea lions to hot-iron branding. Applied Animal Behaviour Science 122, 58–62. Walker, K.A., Horning, M., Mellish, J.E., Weary, D.M., 2009. Behavioural responses of juvenile Steller sea lions to abdominal surgery: developing an assessment of post-operative pain. Applied Animal Behaviour Science 120, 201-207. Waran, N., Best, L., Williams, V., Salinsky, J., Dale, A., Clarke, N., 2007. A preliminary study of behaviour-based indicators of pain in cats. Animal Welfare 16(S), 105-108. Wilson, R.P., McMahon, C.R. 2006. Measuring devices on wild animals: what constitutes acceptable practice? Frontiers in Ecology and the Environment 4, 147-154.  92  CHAPTER 5: Behavioural responses of juvenile Steller sea lions to hot-iron branding 4 5.1 Introduction Animal conservation research often requires that individuals be marked. One marking technique for marine mammals is hot-iron branding. Brands provide a unique mark that can be used to identify individual animals throughout their life. Hot-iron branding has been used to identify cattle and horses for centuries and has been adapted for use in pinnipeds and nondomesticated ungulates. The immediate responses of cattle to hot-iron branding include tail flicking, kicking, and vocalizations (Schwartzkopf-Genswein et al., 1997, 1998) and escapeavoidance responses (Lay et al., 1992). However, no published research has assessed behavioural responses in the days following hot-iron branding in any species. Hot-iron branding of marine mammals has been criticized, resulting in a one-year suspension of hot-iron branding of Steller sea lions in the USA (Dalton, 2005) and an indefinite suspension for elephant seals at Macquarie Island and Hooker’s sea lions in New Zealand (Beausoleil and Mellor, 2007; McMahon et al., 2007). Despite this concern, the effects of branding on pinnipeds have been reported in only five publications, all of which have focused only on physiological responses and survival. Steller sea lions (Eumetopias jubatus) typically experience a general inflammatory response for up to two weeks after hotiron branding (Mellish et al., 2007). In the 12 weeks following hot-iron branding, it was estimated that 1.4 out of every 200 Steller sea lion pup deaths could be attributed to the branding event (Hastings et al., 2009). In harbour seal (Phoca vitulina) pups, 76% of hot-iron 4  A version of the chapter has been published. Walker, K.A., Mellish, J.E., and Weary, D.M. 2010. Behavioural responses of juvenile Steller sea lions to hot-iron branding. Applied Animal Behaviour Science 122:58-62. 93  brands had not healed at 9-10 weeks after branding (Daoust et al., 2006). In Southern elephant seal (Mirounga leonina) pups most brands healed within one year, but brands with more peripheral skin damage had longer healing times (van den Hoff et al., 2004). Longer term monitoring has found no difference in survival between hot-iron branded and flippertagged Southern elephant seals (McMahon et al., 2006). The aim of our study on juvenile Steller sea lions was to describe the post-operative behavioural responses, suggestive of pain, following hot-iron branding using animals that were brought into temporary captivity and branded as a part of a different research project (Mellish et al., 2006). Permit constraints (under National Marine Fisheries Service permit #881-1890) did not allow for experimental treatment groups (e.g. with and without analgesics). We tested the hypotheses that: (1) the behaviour would differ in the days after branding relative to pre-branding baseline measures, and (2) these differences would be most pronounced in the first 24-h period after branding. Specifically, we hypothesized that animals would increase wound-directed behaviours, such as rubbing and scratching the brand, and spend less time lying on their branded side.  5.2 Methods 5.2.1 Study design and animals Research was conducted in collaboration with the Alaska SeaLife Center (ASLC) in Seward, AK, USA. All behavioural research conducted in this study was coordinated with the ASLC’s Transient Juvenile Steller Sea Lion Project (Mellish et al., 2006) where an active hot-iron branding program was already in place.  94  Eleven juvenile Steller sea lions, between 16 and 23 months of age, were captured in Prince William Sound, AK, USA, in August 2007 (three males), February 2008 (one male and one female) and August 2008 (four males and two females) under the Transient Juvenile Steller Sea Lion Project, as described by Mellish et al. (2006). Upon initial handling, symbols were shaved in the animal’s fur on their dorsal side to facilitate identification prior to hotiron branding. The animals were transported to the ASLC and held for up to 12 weeks prior to release. The quarantine facility where the animals were held consisted of four adjoining pools (1 m x 4 m diameter and 3 m x 5 m diameter, 1.5 m deep), with unfiltered seawater from the directly adjacent Resurrection Bay. Each pool was enclosed by 122 m2 of solid metal surface haul-out area. Animals could be housed individually or share access to multiple pools via sliding gates, however they were housed together to the maximum extent possible. All hot-iron branding was performed for the ASLC’s Transient Juvenile study, with no additional handling required for our behavioural observations. No animals were branded simply for the purposes of the current study. All research was conducted under Institutional Animal Care and Use Committee protocols AUP07-009 (ASLC), A07-0342 (UBC), 08-26 (UAF) and approved under National Marine Fisheries Service permit #881-1890.  5.2.2 Study procedures Sea lions were acclimated to the quarantine facility for at least 10 days before they were anaesthetized and hot-iron branded. Animals were masked with 5% isoflurane in 100% oxygen for induction of anaesthesia, after which they were intubated. Anaesthesia was maintained with 1- 3% isoflurane in 100% oxygen delivered via a semiclosed, partially  95  rebreathing circuit. Animals were hot-iron branded with an individually distinctive combination of four numerals. Brands were applied with specially designed stainless steel irons (each 10.2 cm high and 5.1 cm wide) that were heated in a propane-fuelled forge until the irons were cherry red. Each branding iron was then applied to the left shoulder/flank area for 2-7 s each, as previously described by Mellish et al. (2007), with touch-ups to each numeral lasting for 1-4 s each. Branding occurred at the end of the anaesthetic period. The average duration of anaesthesic delivery was 108.8 ± 16.4 min (mean ± S.E.M.; length of anaesthesia varied, as half of the animals had additional body condition and health measurements taken as apart of the larger Transient Juvenile Project, as described by Mellish et al. 2006).  5.2.3 Behavioural observations All behaviours were monitored by live focal animal observations for a total of 6 days: 3 days before branding and 3 days immediately following branding. Days were calculated as 24-h periods, with Day 0 starting immediately following extubation from anaesthesia. With the exception of the day of branding, behavioural sampling occurred six times a day in 10min periods, twice during each period of the day (morning, 09:00 -11:00 h; afternoon, 13:00 15:00 h; and evening, 17:00 -19:00 h). Behaviours were recorded using point-in-time sampling (one scan every min for 10 min). On the day of branding, focal animals were observed after recovering from anaesthesia, which was 1.5 h after extubation from anaesthesia (based on Heath et al., 1997). During this initial observational period, sea lions were monitored continuously with behaviours recorded every min for 1 h. After the first hour of observation, 10 min observations resumed.  96  All observations were made by a trained observer (KAW) hidden from the sea lions’ view behind a plastic blind or via one-way glass depending upon the location of the focal animals. Reliability of this observer’s scores was estimated by comparing scores with another trained observer for 196 scans on six animals. The two observers showed 97% agreement for observed time spent alert and complete agreement for all other behaviours scored. We selected six behaviours that were anticipated to be related to post-operative pain using the following a priori rationale. Wound-directed behaviours: Based on work in other species that have assessed behavioural changes in the days following painful procedures (e.g. Molony et al., 1995), we predicted that sea lions would increase wound-directed grooming and decrease the time spent with pressure on the branded area during periods of lying and sitting. Time spent in daily activities on land or water: Animals have been known to increase or decrease activities after painful procedures depending on the use of the wounded area in performing that behaviour. Based on work in other species (e.g. Flecknell and Liles, 1991), we anticipated a reduction in time spent in locomotion and time spent in the pool. Time spent alert and attentive: Animals exposed to noxious or painful stimuli often increase attentiveness or time spent alert (Roughan and Flecknell, 2004). Therefore, we predicted that sea lions would increase time they were alert after hot-iron branding. Time spent lying down: Based on work in other species after painful procedures (Hemsworth et al., 2009), we predicted that sea lions would decrease time lying after branding. Mutually exclusive behaviours included locomotion, wound-directed grooming and lying time. Locomotion was scored when the sea lion moved on land (i.e. all four limbs are moved at least one body length either by walking on four flippers or by sliding across land) or in water (i.e. swimming – the animal actively propelling itself through the water at least  97  one body length). Wound-directed grooming included scratching, biting and head rubbing the branded area. Sea lions scratched themselves using the front or rear flippers to scrape at their skin. Biting involved the use of teeth to grip or hold the skin, typically in a fast repetitive motion. Head rubbing involved moving the head back and forth with pressure on skin surface. Sea lions were classified as lying down when their bodies were flat on the ground, including dorsal, ventral, and left and right side positioning; the head could be either lifted up or on the ground. Non-mutually exclusive behaviours included time spent in the pool, time on the left (i.e. branded) side and the time spent alert. Time spent in the pool was calculated from activities that occur in the water (i.e. locomotion, floating and foraging). Time spent on the left side was scored when an animal was lying or sitting on land with most of their body weight and pressure on the left side of their body. Time spent alert was scored when an animal was attentive with both eyes open. To determine sample size required power calculations that were computed using preliminary results from August 2007. This analysis indicated that a sample size of between five and 10 individuals would be required to accurately identify the behavioural effects of hot-iron branding. This sampling method was also used by Walker et al. (2009).  5.2.4 Statistical analysis The proportion of time sea lions spent engaged in each of the six behaviours was averaged across the 3 days before branding (Pre-brand), and daily measures were generated for each of the 3 days following surgery (Days 0, 1 and 2). Proportional data were outside the range of 0.3-0.7. Therefore, to condense the distribution and to allow for use in the  98  parametric analyses, all data were arcsine square root transformed (Y =arcsine √p). Mixed model (SAS v9.1) analysis, with a compound symmetry covariance structure, was used to test the effects of hot-iron branding on the six behaviours. The model included the effect of day (Pre-brand, Days 0, 1 and 2). The residuals from the models were tested against the basic assumptions of normality and variance homogeneity, as well as plotted against the predicted values for the model. Specified contrasts were used to compare Pre-brand with each of the 3 days following hot-iron branding (i.e. Pre-brand vs. Day 0, Pre-brand vs. Day 1 and Prebrand vs. Day 2). In all cases, differences were considered to be significant at P ≤ 0.05.  5.3 Results Of the six behaviours measured, four changed after hot-iron branding; locomotion, wound-directed grooming, time in the pool and time spent with pressure on the left (branded) side (Table 5.1).  99  Table 5.1 Least square means and S.E.M. for the proportion of time sea lions (n = 11) spent engaged in six different behaviours before and after hot-iron branding. Means and the S.E.M. are the arcsine square root transformed values (backtransformed means are provided in parentheses). Significant differences for specified contrasts (Pre-brand vs. Day 0, Day 1 and Day 2) are denoted by symbol * (P ≤ 0.05).  Pre-brand  Day 0  Day 1  Day2  Standard error  Locomotion Wound-directed grooming  0.26 (0.07)  0.17 (0.03)*  0.17 (0.03)  0.21 (0.04)  0.04  0.01 (0.00)  0.13 (0.02)*  0.10 (0.01)*  0.06 (0.00)  0.03  Lying down  0.66 (0.37)  0.76 (0.48)  0.78 (0.49)  0.75 (0.46)  0.08  Time in pool  0.43 (0.17)  0.23 (0.05)*  0.34 (0.11)  0.35 (0.12)  0.08  Time on left side  0.28 (0.08)  0.33 (0.10)  0.13 (0.02)*  0.09 (0.01)*  0.05  Time alert  0.86 (0.58)  0.92 (0.64)  0.90 (0.61)  0.78 (0.49)  0.06  Time in locomotion on land and in water decreased from 7% in the Pre-brand period to 3% in Day 0 (F1,30 = 4.40, P = 0.044); by Day 2 values no longer differed from baseline. Sea lions were rarely witnessed scratching, biting or rubbing their left side (area to be branded) in the Pre-brand period. However in the days following hot-iron branding wounddirected grooming increased, with this behaviour occupying approximately 2% of the observation periods on Days 0 and 1 after branding (F1,30 = 10.02, P = 0.004 and F1,30 = 5.71, P = 0.023, respectively). Time spent grooming the branded area returned to Pre-brand levels by Day 2. For comparison, time spent grooming the right side did not differ from Pre-brand levels on Days 0, 1 or 2 (F1,30 = 0.47, P = 0.50, F1,30 = 0.18, P = 0.67 and F1,30 = 0.10, P = 100  0.76, respectively). Similarly, grooming other areas (e.g. head, rump) did not increase on Days 0, 1 or 2 compared with the Pre-brand period (F1,30 = 0.48, P = 0.50, F1,30 = 0.57, P = 0.46 and F1,30 = 1.82, P = 0.19, respectively). No differences were found in lying time. Before branding sea lions spent 17% of the observation period in the pool. Time in the pool declined to 5% on Day 0 (F1,30 = 5.69, P = 0.024), but approached Pre-brand levels on Days 1 and 2. The time sea lions spent with pressure on their left (branded) side showed little change from Pre-brand to Day 0 (N.S.), but decreased to near zero on Days 1 and 2 (F1,30 = 4.28, P = 0.047 and F1,30 = 7.01, P = 0.013, respectively). No differences were found in the time spent alert.  5.4 General discussion In the three days following hot-iron branding sea lions increased wound-directed grooming and spent less time with pressure on their branded side. Increased grooming included head rubbing and scratching the branded area, similar to the wound-directed behaviours in a range of species including calves following dehorning (Faulkner and Weary, 2000) and decapods after exposure to antenna-directed noxious stimuli (Barr et al., 2008). Time spent with pressure on the branded side decreased on Days 1 and 2, possibly due to increased sensitivity to pain, or hyperalgesia, associated with inflammation in the days after injury. Burn injuries in animals that are not treated can lead to sensitivity to previously innocuous stimuli (i.e. allodynia; Pascoe, 2000). The time sea lions spent in activities on land and water decreased following hot-iron branding. Specifically, sea lions decreased the time in the pool and the time they engaged in locomotion during the first 24 h after branding, but both behaviours had returned to Pre-  101  brand levels by 48 h after branding. Pain is also known to affect locomotion (Flecknell and Liles, 1991; Molony and Kent, 1997), especially if the burn location is in an area that is highly mobile or stretched during movement (Hanafiah et al., 2008). Sea lions recovering from abdominal surgery also show reduced locomotion during the 3 days following surgery (Walker et al., 2009). Decreased time in the pool might also have been due to the sensitivity of the brand to salt water. Comparing changes in putative pain-related behaviours before and immediately after hot-iron branding is a first step in understanding pain responses. However, future studies should include branded and unbranded animals, as well as analgesia and anaesthetic groups with and without hot-iron branding (Weary et al., 2006). Reductions of the selected behaviours in response to analgesia would help to support the contention that these are painrelated. Analgesia could involve pre-emptive administration of a local nerve block, the use of orally or intra-muscularly administered analgesics, and the use of local anaesthetic gels and sprays applied after branding. Ideally analgesic protocols will be practical for field use where branding typically occurs and where follow-up may be impossible. Alternative methods of marking sea lions also warrant investigation. It has been suggested that cold-iron branding causes less pain. Hot-iron brands burn through the dermal layers and disrupt the hair follicles preventing new hair growth, whereas cold branding damages the pigment-producing melanocytes but leaves the hair follicles intact allowing for regenerative growth of white hair (Daoust et al., 2006). Studies on cattle show that cold-iron branding causes less pain than hot-iron branding (Schwartzkopf-Genswein et al., 1997). Work on other pinniped species has shown that cold-iron brands heal faster, but hot-iron brands are longer lasting and more legible (Daoust et al., 2006; McMahon et al., 2006).  102  5.5 Conclusion In summary, in the three days following hot-iron branding Steller sea lions spent more time grooming the branded area, less time with pressure on their branded side, less time in the pool and less time in locomotion. These behavioural responses provide a useful foundation for future work monitoring pain following similar procedures in sea lions and other marine mammals and in developing alternative pain management strategies for this procedure.  5.6 Acknowledgments We thank our colleagues in the Animal Welfare Program at the University of British Columbia for many discussions on pain and welfare in marine mammals. We also thank David Mellor for his constructive comments on this manuscript. We are grateful to the veterinary and husbandry departments at the Alaska SeaLife Center for their support. This work was funded in part by the Alaska SeaLife Center, and the Pollock Conservation Cooperative Research Center at the School of Fisheries and Ocean Sciences, University of Alaska Fairbanks, but the findings and conclusions presented by the authors are their own and do not necessarily reflect the views or positions of the funding organizations.  103  5.7 References Barr, S., Laming, P. R., Dick, J. T. A., Elwood, R. W., 2008. Nociception or pain in a decapod crustacean? Anim. Behav. 75, 745-751. Beausoleil, N. J., Mellor, D. J., 2007. Investigator responsibilities and animal welfare issues raised by hot branding of pinnipeds. Aust. Vet. J. 85, 484-485. Dalton, R., 2005. Animal-rights group sues over ‘disturbing’ work on sea lions. Nature 436, 315. Daoust, P., Fowler, G. M., Stobo, W. T., 2006. Comparison of the healing process in hot and cold brands applied to harbour seal pups (Phoca vitulina). Wildl. Res. 33, 361-372. Faulkner, P. M., Weary, D. M., 2000. Reducing pain after dehorning in dairy calves. J. Dairy Sci. 83, 2037–2041. Flecknell, P. A., Liles, J. H., 1991. The effects of surgical procedures, halothane anaesthesia and nalbuphine on the locomotor activity and food and water consumption in rats. Lab. Anim. 25, 50-60. Hanafiah, Z., Potparic, O., Fernandez, T., 2008. Addressing pain in burn injury. Curr. Anaesth. Crit. Care 19, 287-292. Hastings, K.K., Gelatt, T.S., King, J.C., 2009. Postbranding survival of Steller sea lion pups at Lowrie Island in Southeast Alaska. J. Wildl. Manage. 73, 1040-1051. Heath, R. B., DeLong, R., Jameson, V., Bradley, D., Spraker, T., 1997. Isoflurane anesthesia in free-ranging sea lion pups. J. Wildl. Dis. 33, 206-210. Hemsworth, P. H., Barnett, J. L., Karlen, G. M., Fisher, A. D. Butler, K. L., Arnold, N.A., 2009. Effects of mulesing and alternative procedures to mulesing on the behaviour and physiology of lambs. Appl. Anim. Behav. Sci. 117, 20-27. Lay, D. C., Friend, T. H., Bowers, C. L., Grissom, K. K., Jenkins, O. C. A., 1992. A comparative physiological and behavioral study of cold and hot-iron branding using dairy cows. J. Anim. Sci. 70, 1121-1125. McMahon, C. R., Burton, H. R., van den Hoff, J., Woods, R., Bradshaw, C. J., 2006. Assessing hot-iron and cryo-branding for permanently marking southern elephant seals. J. Wildl. Manage. 70, 1484-1489. McMahon, C. R., Bradshaw, C. J. A., Hays, G. C., 2007. Applying the heat to research techniques for species conservation. Conserv. Biol. 21, 271-273.  104  Mellish, J., Calkins, D., Christen, D., Horning, M., Rea, L., Atkinson, S., 2006. Temporary captivity as a research tool: comprehensive study of wild pinnipeds under controlled conditions. Aquat. Mamm. 32, 58-65. Mellish, J., Hennen, D., Thomton, J., Petrauskas, L., Atkinson, S., Calkins, D., 2007. Permanent marking in an endangered species: physiological response to hot branding in Steller sea lions (Eumetopias jubatus). Wildl. Res. 34, 1-6. Molony, V., Kent, J. E., Robertson, I. S., 1995. Assessment of acute and chronic pain after different methods of castration of calves. Appl. Anim. Behav. Sci. 46, 33-48. Molony, V., Kent, J. E., 1997. Assessment of acute pain in farm animals using behavioural and physiological measurements. J. Anim. Sci. 75, 266-272. Pascoe, P. J., 2000. Problems of pain management, in: Flecknell, P., Waterman-Pearson, A., (Eds.), Pain Management in Animals, Harcourt Publishers Limited, London, pp. 161-177. Roughan, J.V., Flecknell, P.A., 2004. Behaviour-based assessment of the duration of laparotomy- induced abdominal pain and the analgesic effects of carprofen and buprenorphine in rats. Behav. Pharm. 15, 461-472. Schwartzkopf-Genswein, K. S., Stookey, J. M., dePassille, A. M., Rushen, J., 1997. Comparison of hot-iron and freeze branding on cortisol levels and pain sensitivity in beef cattle. Can. J. Anim. Sci. 77, 369-374. Schwartzkopf-Genswein, K. S., Stookey, J. M., Crowe, T. G., Genswein, B. M. A., 1998. Comparison of image analysis, exertion force, and behaviour measurements for use in the assessment of beef cattle responses to hot-iron and freeze branding. J. Anim .Sci. 76, 972979. van den Hoff, J., Sumner, M. D., Field, I. C., Bradshaw, C. J. A., Burton, H. R., McMahon, C. R., 2004. Temporal changes in the quality of hot-iron brands on elephant seal (Mirounga leonine) pups. Wildl. Res. 31, 619-629. Walker, K. A., Horning, M., Mellish, J. E., Weary, D. M., 2009. Behavioural responses of juvenile Steller sea lions to abdominal surgery: developing an assessment of post-operative pain. Appl. Anim. Behav. Sci. 120, 201-207. Weary, D. M., Neil, L., Flower, F. C., Fraser, D., 2006. Identifying and preventing pain in animals. Appl. Anim. Behav. Sci. 100, 64-76.  105  CHAPTER 6: Effects of hot-iron branding on heart rate, breathing rate and behaviour of anaesthetized Steller sea lions 5  6.1 Introduction The response of animals to noxious stimuli can be studied using changes in behavioural and physiological parameters (Livingston and Chambers, 2000). Behavioural responses will vary among species, but may include pain-specific behaviours that occur during the application of noxious stimuli. For example, in cattle immediate responses to hotiron branding include tail flicking, kicking, vocalizations (Schwartzkopf-Genswein et al., 1997, 1998) and escape-avoidance responses (Lay et al., 1992). Physiological responses to noxious stimuli include changes in the autonomic nervous system, including activation of the hypothalamic-pituitary-adrenal and the sympathetic nervous system (SNS). Activation of the SNS affects the visceral responses by increasing heart and breathing rates and releasing catecholamines (epinephrine and norepinephrine). Electrocardiograms (ECG) allow for the measurements of heart activity. Heart rate varies from beat to beat during normal sinus rhythm and is increased by activation of the SNS, especially the release of epinephrine and norepinephrine (Task Force, 1996). Changes in heart rate have been measured after the application of noxious stimuli in a variety of animals (e.g., in cattle, Lay et al., 1992; lambs, Peers et al., 2002; mice, Arras et al., 2007; goats, Alvarez et al., 2009).  5  A version of the chapter will be submitted for publication. Walker, K.A., Mellish, J.E. and Weary, D.M. 2010. Effects of hot-iron branding on heart rate, breathing rate and behaviour of anaesthesized Steller sea lions. 106  Hot-iron branding, while considered a noxious stimulus, is a common procedure for wildlife biologists to allow for long-term tracking of seals and sea lions. Depending on the species and particular field situation, hot-iron branding may occur with and without general gas anaesthesia. If branding occurs with anaesthesia, it is common for inhalant anaesthestics, such as isoflurane, to be used (Haulena and Heath, 2001). The aim of general anaesthesia is to produce unconsciousness, amnesia and to provide immobilization during noxious stimulation (Antognini et al., 2005). Whelan and Flecknell (1992) define a surgical plane of general anaesthesia as “being in the state in which an animal is immobile, unaware of the procedure being performed and has an attenuated stress response”. A variety of criteria, often in combination, are used to assess depth of anaesthesia (Whelan and Flecknell, 1992; Antognini et al., 2005). Autonomic parameters, including heart rate, blood pressure and breathing rate provide insight into the physiological state of the animal and can be used to help monitor anaesthestic depth. In marine mammals, assessment of anaesthetic depth typically relies on the animal’s reflex responses, jaw tone, respiratory character, and response to various stimuli (Haulena and Heath, 2001). The physiological (Mellish et al., 2007) and behavioural (Walker et al., 2010) responses of sea lions to hot-iron branding have been monitored in the days following branding, but to our knowledge no previous work has assessed the immediate response of sea lions to branding. In addition, few studies have assessed responses of anaesthetized animals to noxious stimuli (e.g., lamb castration, Johnson et al., 2005), but to our knowledge no previous work has assessed responses in anaesthetized marine mammals. The aim this study was to assess heart rate, breathing rate and behaviour in anaesthetized Steller sea lions during hot-iron branding.  107  6.2 Methods 6.2.1 Study animals Research was conducted in collaboration with the Alaska SeaLife Center (ASLC) in Seward, AK, USA. This study utilized animals that were branded as part of the ASLC’s Transient Juvenile Steller Sea Lion Project (Mellish et al., 2006). No animals were branded simply for the purposes of the current study. Twelve juvenile sea lions (transient juveniles: TJs) 16 to 23 months of age were captured in Prince William Sound, AK, USA, as described by Mellish et al. (2006), with 6 captured in August 2008 and 6 in May 2009. Animals were held in a specialized quarantine facility for up to 12 weeks. The facility consisted of four adjoining pools (1 x 4 m diameter and 3 x 5 m diameter, 1.5 m deep) that contained unfiltered seawater, enclosed by 122 m2 of solid metal surface haul-out area. Sea lions were acclimated to the quarantine facility for at least 10 days before hot-iron branding. On the day of branding, sea lion’s weighed on average 107 ± 22 kg (mean ± S.D.).  6.2.2 Design Hot-iron brands consisted of a unique combination including one symbol (an “=” sign) and three numerals. This combination allowed for an individually distinctive marking that identified the research group and the age of the animal at branding, consistent with the requirements of a nationally collaborative branding database (i.e., Alaska SeaLife Center, National Marine Mammal Laboratory, Alaska Department of Fish and Game). Brands were applied with specially designed stainless steel irons, 10.2 cm high and 5.1 cm wide, heated in  108  a propane-fuelled forge until the irons were cherry red. Each branding iron was then applied to the left shoulder/flank area for 2 to 7 s each, as previously described by Mellish et al. (2007). Touch-ups to the brand (i.e., iron re-applied to the animal’s skin) occurred on all animals, ranging from 3 to 8 touch-up events per animal, with each touch-up lasting for 1 to 4 s. All animals underwent both a sham and a hot-iron branding procedure while under gas anaesthesia. Data were recorded for all 12 animals during 4 periods (in order): (1) Baseline, 5 min, (2) Sham, 1 min, (3) Brand, 2.7 ± 0.4 min (mean ± S.E.M.) and (4) Postbrand, 5 min. The Brand period was calculated as the time a hot iron first touched the animal and ending when the last iron was finally removed. Physiological (heart rate and breathing rate) and behavioural measures (trembling/shaking, head/neck/shoulder movement) were recorded continuously during each of the 4 periods. The order of the 4 periods was followed for all but 2 animals: TJ 50 had an abscess wound drained after the Baseline period, with this manipulation ending approximately 3 min prior to the Sham period, and TJ 54 had a previously infected biopsy wound washed 30 s after the Sham period ended, with this manipulation ending 4 min prior to Branding. In addition, TJ 54 had two blood samples drawn during the Baseline period and ECG were recorded. Sham branding occurred 3.7 ± 0.7 min (mean ± S.E.M) after the Baseline period and 4.6 ± 0.7 min before the Brand period. During sham branding, an unheated branding iron was placed for 2 to 4 s on the animal in the same location as the future placement of the hot-iron brand, on the animals left side flank. The sham branding allowed us to separate the animal’s responses to the hot-iron branding procedure from the animal’s response to anaesthesia,  109  handling, and pressure of the brand iron. The same person performed sham and hot-iron branding. All sea lions were masked with 5% isoflurane in 100% oxygen, after which they were intubated for the remainder of the procedure. Anaesthesia was maintained with 1-3% isoflurane in 100% oxygen delivered via a semi-closed, partial re-breathing circuit. The average duration of anaesthesia was 60 ± 5 min (mean ± S.E.M). Hot-iron branding occurred approximately 54 ± 6 min after sea lions were masked with isoflurane. Depth of anaesthesia was determined by reflex responses, jaw tone, respiratory character, and response to various stimuli including noise, ear and tail tugs, nose tap, and rectal thermometer insertion, as per Haulena and Heath (2001). The attending veterinarian monitored depth of anaesthesia on a 0 to 5 scale (0 - fully awake; 1 – reactive, voluntary movements; 2 – reactive, no voluntary movements, increased respiration; 3 – non-reactive, decreased respiration; 4 – apnea; 5 – cardiac and respiratory depression) and attempted to maintain anaesthetic level at 3 during all periods.  6.2.3 Physiological measures An electrocardiograph (ECG) was used to monitor changes in heart rate using the SmartHeart™ Vet program and stored for later analysis. Three leads from the ECG were attached via alligator clips to the trailing edges of the right and left front flipper and the left rear flipper, near the insertion point to the trunk, approximately 15 min before baseline recording began. Due to technical difficulties with the ECG, we are missing data for the 5min Post-brand period for one animal. Inspection of the ECG signals allowed us to visually identify QRS complexes, and to measure the distance between adjacent R-R peaks. Artefacts,  110  ectopic cardiac beats, and segments of data that contained more than 10% artefacts (the Brand period for two animals) were removed from the analysis. The resulting R peaks were then used to produce a complete R-R interval time series, from which the mean instantaneous heart rate was calculated. Breathing rate was measured from concurrent video by two trained observers (KAW and VK) counting the total inhalations per minute in each of the four periods. The two observers showed 100% agreement.  6.2.4 Behavioural measures Trembling (i.e., rhythmic shaking) and the movement of the head, neck or shoulder were recorded by live observation as either present or absent (regardless of duration) during each of the four observation periods.  6.2.5 Statistical analyses Data were analysed using a mixed model (SAS v9.1), with an autoregressive covariance structure, and subject (sea lion) specified as a random effect. The model included period (Baseline, Sham, Brand, Post-brand) as the repeated measure. Residuals from the model were tested against the basic assumptions of normality and variance homogeneity, and plotted against the predicted values for the model. Specified contrasts were used to compare pre-branding baseline with the other 3 periods (i.e., Baseline vs. Sham, Baseline vs. Brand, and Baseline vs. Post-brand). In all cases, differences were considered to be significant at P ≤ 0.05.  111  6.3 Results Changes in heart rate over the four periods are illustrated for all 12 sea lions (Fig. 6.1). Overall, heart rate showed little change from Baseline to the Sham period, averaging 78.3 ± 2.4 bpm and 79.5 ± 2.5 bpm respectively (F1,30 = 2.09, P = 0.159). Heart rate increased over Baseline for all 12 animals during Branding, averaging 85.6 ± 2.5 bpm (F1,30 = 26.94, P < 0.001). A gradual decline was noted in the Post period, but heart rate remained elevated over Baseline observations (84.7 ± 2.5 bpm; F1,30 = 14.91, P = 0.006).  112  TJ 43  TJ 44  90  90 85  85  B S  P  80  80  75  75  70  70  B  S  65  65 0  400  600  800  1000  0  95  TJ 45  95  Heart Rate (bpm)  200  200  400  600  TJ 46  800  1000  P  P S  90  90  B  85  85  80  80  75  75  B S \  70 0  200  400  600  800  1000  70 0  200  400  600  800  1000  600  800  1000  P TJ 47  100  TJ 48 S  P  100  B  95  95  90  S  B  90  85  85  80  80  75  75  70 0  200  400  600  800  1000  70 0  Time (sec)  113  200  400  TJ 50  TJ 51  85 Abscess wound washing  80  P  3 min break  Baseline  100 S  75  P  105  B  B  95  70  90  65  85 80  60 0  200  400  600  800  0  1000  200  400  600  800  1000  P P  Heart Rate (bpm)  TJ 52  TJ 53  90  95  85  S  90 B  80  85  75  80  70  75  65 0  200  400  600  Biopsy wound wash, then 4 min break  TJ 54  115 Blood draw  110 Blood draw  105  800  1000  S  B  70 0  200  400  800  600  800  1000  P  TJ 55  P  600  95 S  B  S  90  B  85  100 80  95  75  90 85 0  200  400  600  800  1000  70 0  200  400  1000  Time (sec)  Figure 6.1 Instantaneous heart rates of 12 individual sea lions during four periods. Arrows represent beginning of a period; S – Sham brand, B – Brand, and P – Post-brand. Graphs begin with the 5 min Baseline period. Note, TJ 43 does not have a Post-brand period and TJ 51 had the Sham brand period occur before the Baseline period. Abscess wound cleaning is shown for TJ 50 and biopsy wound and blood draws for TJ 54. 114 114  There was no difference in average breathing rate from Baseline to Sham (F1,32 = 0.06, P = 0.801); sea lions took on average about 2.5 breaths per minute during both periods (Fig. 6.2). Breathing rate increased more than three fold from the Baseline to the Brand period (F1,32 = 24.96, P < 0.001) with animals taking approximately 9 breaths per minute, but returned to Baseline levels in the Post-brand period (F1,32 = 0.94, P = 0.340).  Breathing rate (bpm)  10 8 6 4 2 0 Baseline  Sham  Brand  Post-brand  Figure 6.2. Average breathing rate (breaths per minute) for sea lions during the 4 experimental periods. Data are presented as least square means ± S.E.M.  115  One animal was observed trembling during the Baseline period. No animals exhibited trembling or body movements during Sham branding. In the Brand period, 5 animals were observed trembling and moving their head or shoulders, 1 was observed trembling only and 1 was observed only moving their heads and shoulders; 5 animals showed no behavioural response. In the Post-branding period, 2 of the animals that were observed trembling during Branding continued this behaviour.  6.4 Discussion Autonomic responses to painful stimuli, including increased heart rate, have been witnessed in a variety of animals (e.g., branding, Lay et al., 1992; castration and tail-docking, Peers et al., 2002). In this study, sea lions had increased heart and breathing rates during branding relative to the Baseline and Sham periods. Behavioural responses included trembling and head and shoulder movements, with half of the animals observed responding behaviourally to branding. One animal was witnessed trembling during Baseline, perhaps due to lowered core body temperature resulting from anaesthesia. Movements alone do not indicate that animals are under-anaesthesized; movements may instead reflect simple reflex withdrawals while the animal remains unconscious (Antognini et al., 2005). Half of the sea lions in our study exhibited a behavioural response, and all animals had increased heart and breathing rates. Isoflurane is thought to provide ‘excellent muscle relaxation’ (Muir et al., 2000), but has ‘poor analgesic properties’ (Yentis et al., 1996). Trembling, body movements, and increased heart and breathing rate noted in this study, indicate that isoflurane anaesthesia as administered may not be sufficient to maintain a surgical plane of anaesthesia in sea lions  116  during branding (Haskins, 2007). A surgical plane of anaesthesia is required to ensure that the pain experienced by the animal is eliminated (NRC, 2009), but there are also risks associated with a higher plane of anaesthesia (e.g., depression of vital organ function). All anaesthesia involves a balancing of these risks, but exactly how this balance is achieved varies depending upon how much value the anaesthetist places on the risk of pain versus the risk of complications (Flecknell, 2009). Anaesthetists working on marine mammals may be more likely to err on the side of under treatment due to the complications that may arise from dive reflexes, or due to the desire to have a quick anaesthetic recovery. If a lighter plane of anaesthesia is used then the administration analgesics, such as a local nerve block or opioid, may help reduce pain responses during the procedure (Antognini et al., 2005). Analgesics work by limiting the amount of nociceptive input to the central nervous system. The use of pre-emptive analgesia has also been shown to reduce postoperative pain responses by reducing sensitization caused by damaged tissues. A local nerve block and the administration of general analgesic agents via an oral or intra-muscular route are recommendations for pre-emptive analgesia, while local anaesthetic gels and sprays applied after branding can be used post-operatively. Research supports the use of combined pre- and post-operative analgesia (Dobromylskyj et al., 2000) and may help to reduce both the intra- and post-operative responses witnessed in sea lions in response to hot-iron branding. Concerns have been raised about the pain associated with hot-iron branding (Beausoleil and Mellor, 2007); however, to date only one other study has assessed the pain associated with branding in sea lions (Walker et al., 2010). Sea lions responded behaviourally in the 3 d after branding by spending more time grooming their branded area, less time with  117  pressure on their branded side, and less time in the pool and in locomotion, suggesting that alternative analgesia protocols be investigated. The results of the current study indicate that sea lions experienced nociception, possibly even pain, at the time of branding and that the animals were at a light level of anaesthesia (Haskins, 2007). A more complete understanding of the degree of pain experienced would require techniques other than those presented here. For example, the electroencephalograph (EEG) can be used to measure the cortical response to pain stimulus and might also provide information about pain perception in sea lions (Murrell and Johnson, 2006). Our research would also have benefited from the measurement of alveolar gas concentrations, via an airway gas analyzer. This would have allowed the veterinarian to provide similar end tidal concentrations for each animal, helping to ensure that all sea lions were at a similar plane of anaesthesia. It has been suggested that changes in beat-to-beat variability, known as heart rate variability (HRV), allows for greater interpretation of autonomic nervous system responses. Research has begun to use HRV as an indicator of pain (Rietmann et al., 2004; Arras et al., 2007; Stewart et al., 2008). Heart rate cannot be used to assess sympathovagal regulation due to the difficulties in distinguishing a reduction in vagal activity from an increase in sympathetic activity, or a combination of both (von Borell et al., 2007). Unfortunately, HRV measurements were not possible in the current study as HRV analysis requires comparisons of equal periods of 5 min or greater. The responses during branding can be compared with other invasive procedures performed on the sea lions. For example, TJ 54 had blood samples taken during the Baseline  118  period (see Fig. 6.1 for timing) with no apparent heart, breathing or behavioural responses noted. Two animals had wounds that were treated during anaesthesia and their responses were recorded. TJ 50 had a flipper abscess wound drained and cleaned out, during which heart rate averaged 74 bpm and breathing rate 12 bpm. This can be compared the Brand period where the animal had a heart rate of 71 bpm and a breathing rate of 5.5 bpm. TJ 54 had an infected biopsy wound that required washing. A mean heart rate of 98 bpm was recorded during wound cleaning compared with 105 bpm during branding; breathing rate data were not collected during wound cleaning. No behaviours were noted for either animal. These results suggest that TJ 50 was most likely experiencing break through pain during wound treatment, but TJ 54 was not. The results of the current study and Walker et al. (2010) suggest that sea lions are experiencing pain both during and after hot-iron branding. Results to date suggest that branding does not affect survival (McMahon et al. 2006; Hastings et al., 2009), but branding does produce a generalized inflammatory response based on various blood parameters, with levels returning to baseline 7-8 wk post-branding (Mellish et al., 2007). In some marine mammal species, brands may take up to one year to heal (van den Hoff et al., 2004), but reasons for slow and variable rates of healing are not known. Ultimately, the welfare consequences of hot-iron branding need to be weighed against the value of any information gained from this type of marking by wildlife researchers (Beausoleil and Mellor, 2007).  119  6.5 Conclusions Autonomic responses were observed in all anaesthetized sea lions at the time of hotiron branding, while behavioural responses were witnessed in half of the animals. These results indicate that isoflurane anaesthesia alone, as carried out in the current study, does not provide sufficient anaesthesia to eliminate the sea lion’s immediate responses to branding. Future investigations into the use of peri-operative analgesia may help in reducing these responses.  6.6 Acknowledgments We thank Vanessa Kuhnen for her help measuring ECG segments and Markus Horning for lending us the ECG equipment. We are grateful to the husbandry and veterinary departments at the Alaska SeaLife Center for their support. We are also grateful to our colleagues in the Animal Welfare Program at the University of British Columbia for many discussions on pain and welfare in marine mammals. This study was supported by the Alaska SeaLife Center, and by UBC’s Animal Welfare Program and its donors listed at http://www.landfood.ubc.ca/animalwelfare.  120  6.7 References Alvarez, L., A.R. Nava, A. Ramirez, E. Ramirez and J. Gutierrez 2009. Physiological and behavioural alterations in disbudded goat kids with and without local anaesthesia. Applied Animal Behaviour Science 117: 190-196. Antognini, J., L. Barter and E. Carstens. 2005 Movement as an index of anesthetic depth in humans and experimental animals. Comparative Medicine 55: 413-418. Arras, M., A. Rettich, P. Cinelli, H. P. Kasermann and K. Burki. 2007. Assessment of postlaparotomy pain in laboratory mice by telemetric recording of heart rate and heart rate variability. BMC Veterinary Research 3: doi:10.1186/1746-6148-3-16. Beausoleil, N. J. and D.J. Mellor. 2007. Investigator responsibilities and animal welfare issues raised by hot branding of pinnipeds. Australian Veterinary Journal 85: 484-485. Dobromylskyj, P., Flecknell, P.A., Lascelles, B.D., Pascoe, P.J., Taylor, P., WatermanPearson, A. 2001. Management of postoperative and other acute pain. Pages 81-145 in Pain Management in Animals. Flecknell, P., Waterman-Pearson, A. (Eds.) Harcourt Publishers Limited, London, UK. Flecknell, P. 2009. Laboratory Animal Anaesthesia, Third Edition. Academic Press, London, UK. 300 pp. Haskins, S.C. 2007. Monitoring anesthetized patients. Pages 533-560 in Lumb and Jones’ Veterinary Anesthesia and Analgesia, 4th Edition. Tranquilli, W.J., Thurmon, J.C. and Grimm, K.A. (Eds.) Blackwell Published, Iowa, USA. Haulena, M. and R.B. Heath. 2001. Marine mammal anesthesia. Pages 655 – 688 in the CRC Handbook of Marine Mammal Medicine. Dierauf, L.M. and Gulland, F.M.D. (Eds.) CRC Press, Florida, USA. Johnson, C.B., K.J. Stafford, S.P. Sylvester, R.N. Ward, S. Mitchinson and D.J. Mellor. 2005. Effects of age on the electroencephalographic response to castration in lambs anaesthetised using halothane in oxygen. New Zealand Veterinary Journal 53: 433-437. Lay, D.C., T.H. Friend, C.L. Bowers, K.K. Grissom and O.C. Jenkins. 1992. A comparative physiological and behavioral study of freeze and hot-iron branding using dairy cows. Journal of Animal Science 70: 1121-1125. Livingston, A. and P. Chambers. 2000. Physiology of pain. Pages 9-19 in Pain Management in Animals. Flecknell P.A. and Waterman- Pearson A.E. (Eds) Harcourt Publishers Limited, London, UK.  121  Mellish, J., D. Calkins, D. Christen, M. Horning, L. Rea and S. Atkinson. 2006. Temporary captivity as a research tool: comprehensive study of wild pinnipeds under controlled conditions. Aquatic Mammals 32: 58-65. Mellish, J., Hennen, D., Thomton, J., Petrauskas, L., Atkinson, S., Calkins, D. 2007. Permanent marking in an endangered species: physiological response to hot branding in Steller sea lions (Eumetopias jubatus). Wildlife Research 34: 1-6. Muir, W. 1994. Balanced anaesthesia: new emphasis on an old idea. Journal of Veterinary Anaesthesiology 21: 9-11. Muir, W.W., J.A.E. Hubbell, R.T. Skarda and R.M. Bednarski. 2000. Pharmacology of Inhalation Anesthetic Drugs. Pages 164-181 in Handbook of Veterinary Anesthesia, 3rd ed, Mosby, St. Louis. Murrell, J.C. and C.B. Johnson. 2006. Neurophysiological techniques to assess pain in animals. Journal of Veterinary Pharmacology and Therapeutics 29: 325-335. National Research Council, NRC. 2009. Recognition and Alleviation of Pain in Laboratory Animals. The National Academies Press, Washington, D.C. Peers, A., D.J. Mellor, E.M. Wintour and M. Dodic. 2002. Blood pressure, heart rate, hormonal and other acute responses to rubber-ring castration and tail docking of lambs. New Zealand Veterinary Journal 50: 56–62. Rietmann, T.R., M. Stauffacher, P. Bernasconi, J.A. Auer and M.A. Weishaupt. 2004. The association between heart rate, heart rate variability, endocrine and behavioural pain measures in horses suffering from laminitis. Journal of Veterinary Medicine 51: 218-225. Schwartzkopf-Genswein, K. S., J.M. Stookey, A.M. dePassille and J. Rushen. 1997. Comparison of hot-iron and freeze branding on cortisol levels and pain sensitivity in beef cattle. Canadian Journal of Animal Science 77: 369-374. Schwartzkopf-Genswein, K. S., J.M. Stookey, T.G. Crowe and B.M.A. Genswein. 1998. Comparison of image analysis, exertion force, and behaviour measurements for use in the assessment of beef cattle responses to hot-iron and freeze branding. Journal of Animal .Science 76: 972-979. Stewart, M., K.J. Stafford, S.K. Dowling, A.L. Schaefer and J.R. Webster. 2008. Eye temperature and heart rate variability of calves disbudded with or without local anaesthetic. Physiology & Behavior 93: 789-797. Task Force of the European Society of Cardiology and the North American Society of Pacing and Electrophysiology. 1996. Heart rate variability: standards of measurement, physiological interpretation and clinical use. Circulation 93:1043-65.  122  Von Borell, E., J. Langbein, G. Depres, S. Hansen, C. Leterrier, J. Marchant-Forde, R. Marchant-Forde, M. Minero, E. Mohr, A. Prunier, D. Valance and I. Veisser. 2007. Heart rate variability as a measure of autonomic regulation of cardiac activity for assessing stress and welfare in farm animals – A review. Physiology & Behaviour 92: 293-316. Walker, K.A., J.E. Mellish and D.M. Weary. 2010. Behavioural responses of juvenile Steller sea lions to hot-iron branding. Applied Animal Behaviour Science 122: 58-62. Whelan, G. and P.A. Flecknell. 1992. The assessment of depth of anaesthesia in animals and man. Laboratory Animals 26: 153-162. Yentis, S.M., N.P. Hirsch, and G.B. Smith. 1996. Encyclopedia of Anesthesia Practice. Ed. Feeley, T.W. Butterworth-Heinemann, Newton.  123  CHAPTER 7: General discussion  The use of more invasive marking techniques, such as hot-iron branding, is falling under public scrutiny (McMahon, 2007). The concerns over hot-iron branding, combined with the lack of information on the individual-level effects on the animal, led to a successful lawsuit by the Humane Society of the United States (Dalton, 2005). This lawsuit resulted in a one-year suspension of hot-iron branding in the USA. Indefinite suspensions have occurred for elephant seals at Macquarie Island and Hooker’s sea lions in New Zealand (Beausoleil and Mellor, 2007; McMahon et al., 2007). Research involving invasive marking techniques will likely continue to be criticized by the public unless the methods can be demonstrated to have little impact on the animal. Guidelines for field research state that markings should not cause pain or distress (Friend et al., 1994; Nietfeld et al., 1994; Murray and Fuller, 2000). Wildlife care and use guidelines, such as those of the Canadian Council on Animal Care, recommend that researchers use analgesia and anaesthetics for invasive procedures (CCAC, 2003). If pain is present due to a research-related injury, then researchers should modify their procedures to reduce the animals’ suffering (CCAC, 2003). Pain can also affect aspects of an animal’s normal functioning; therefore reducing animal pain at the time of marking or tagging has the potential to improve the quality of the scientific data. However, pain assessments are rarely included or described in field research (see Chapter 2). The recognition and alleviation of pain is often left to the attending veterinarian (if present during handling procedures). This is not to suggest that field researchers are  124  oblivious to the potential for pain in their study animals, but assessment and treatment may be hampered by a lack of appropriate methodology. Studies under controlled conditions allow for the use of appropriate controls and can help in developing methods of pain assessment. The temporary captivity research model of the Transient Juvenile Steller Sea Lion Project at the Alaska SeaLife Center, Seward, Alaska, USA (Mellish et al., 2006), provided an ideal setting to address the issue of pain surrounding abdominal surgery and hot-iron branding.  7.1 Welfare concerns associated with abdominal surgery Abdominal surgery for the implantation of telemetry devices, such as the LHX tags used for sea lions, has allowed researchers to monitor animals for longer periods of time compared with externally placed devices; however, no research had assessed the postoperative pain associated with abdominal surgery. The objective of Chapter 3 was to describe the specific behavioural responses that occur in juvenile Steller sea lions following abdominal surgery. Sea lions displayed a variety of behaviour after the abdominal surgery required for the implantation of LHX tags, including standing and back arch, which were not seen before surgery. Sea lions may perform these behaviours to promote recovery of the wound and reduce stimulating the area of the injury. Hyperalgesia (increased sensitivity to pain) can occur in response to inflammatory pain and may explain why back arch and standing persist at 2 wk after surgery. Flunixin meglumine administered only once immediately after surgery was most likely not fully effective in controlling post-operative pain; investigations of alternative analgesic methods for this procedure are warranted. Therefore, Chapter 4 was designed to test carprofen as an alternative to flunixin for 125  controlling post-operative pain. All seven behaviours changed after surgery regardless of NSAID treatment; only one behaviour was mildly affected by analgesic treatment. These results suggested that neither treatment was effective in controlling pain in the days following abdominal surgery. The pharmacokinetics of the analgesics used in these experiments have not been tested on sea lions. The choice of analgesia, route of administration and dosages were chosen based on previous work in sea lions (flunixin is the current analgesia used after this abdominal surgery in sea lions: Horning et al., 2008; carprofen is used clinically with captive Steller sea lions at the ASLC) and were based on data extrapolated from other species and the manufacturer’s recommendations for canines. Therefore, it is possible that the dosages administered were too low or the drugs are ineffective for controlling pain in sea lions. It is also possible that the use of fish to deliver the carprofen dose may have slowed absorption (Ray et al., 1979; Nolan, 2000) and perhaps reduced the effect of the drug.  7.1.1 Future abdominal surgery research Given the shortcomings of the treatments used to date, multimodal analgesic therapies, including administration of pre-, intra-, and post-operative analgesia, may provide greater analgesic effect and fewer adverse effects (Dobromylskyj et al., 2000; VinuelaFernandez et al., 2007). The standard approach to treating post-operative pain after sea lion abdominal surgery has been to give analgesics during or immediately after surgery. Administration of treatment before surgery may influence the post-operative pain responses by reducing the firing of nociceptors and thus limiting the hyperalgesia induced by oversensitization from damaged tissues (Dobromylskyj et al., 2000). Combined pre- and  126  post-operative analgesic treatment may also be more effective than pre- or post-operative treatment alone. Increased duration and frequency of post-operative NSAID administration or the administration of a different class of NSAID may be effective, but there is no evidence that NSAIDs alone control pain after abdominal surgery. Opioids are used for moderate to severe pain (Nolan, 2000) and when used in combination with NSAIDs have been shown to be more effective than the use of NSAIDs or opioids alone (e.g., ovariohysterectomy surgery in dogs: Slingsby and Waterman-Pearson, 2001). An ideal pain management study would involve four treatment groups: with and without the painful stimuli and with and without the use of analgesics. However, withholding analgesics after a painful procedure is not in the best interest of the animal. I suggest instead that the current analgesic protocol (flunixin meglumine as described in Chapter 3) be used as a control. With this in mind, I propose the following experimental treatment groups be tested to help reduce pain in captive sea lions after abdominal surgery: (1) a Control group: postoperative one time IM administration of flunixin meglumine, (2) an NSAID/Opioid group: pre- or intra- operative IM administration of an opioid such as morphine or fentanyl, postoperative IM administration of carprofen or ketoprofen, followed by 3x daily doses of carprofen or ketoprofen administered orally through placement in fish for 5 d, and (3) an Opioid group: opioid, such as morphine or fentanyl, administered pre-, intra- and postoperatively, followed by daily doses morphine or fentanyl administered orally through placement in fish for 5 d. Additionally, longer monitoring of the sea lions after surgery is needed to determine when, and if, the behavioural responses to surgery dissipate.  127  Chapter 3 included two treatment groups: abdominal surgery with no branding and abdominal surgery with branding. This design did not allow us to separate the effects of branding. Ideally, two more treatment groups (no abdominal surgery and branding, and no abdominal surgery and no branding) would allow for stronger comparisons of the cumulative effects of these procedures on the animals. The current experiment was also confounded by an alteration in the line block protocol, such that animals in August 2007 did not receive a local block whereas the February 2008 group received a local anaesthesic line block. This added to the already complicated design of cumulative effects (branding with surgery, and surgery only). Future research could benefit from adjustments to the data collections methods. Frequency of occurrence for behaviours such as grooming, vocalizations and number of times the animals entered and exited the pool may provide more insight into the effects of abdominal surgery.  7.2 Welfare concerns associated with hot-iron branding Hot-iron branding has provided wildlife researchers with a tool to permanently mark wildlife (see Chapter 5 for full description), but it comes at a cost to the animal. A number of studies on pinnipeds have assessed branding (e.g., Daoust et al., 2006, McMahon et al., 2006) and the subsequent readability of brands (van den Hoff et al., 2004), however research on the individual effects on the animal has been largely limited to a histological examination (Daoust et al., 2006) and a study of physiological parameters (Mellish et al., 2007). No prior research had assessed pain responses of sea lions to hot-iron branding; in fact, no published research has evaluated behavioural responses in the days after hot-iron  128  branding in any species. Chapter 5 assessed Steller sea lion behaviour in the 3 d after hot-iron branding and found behavioural changes for up to 72 h after branding. These results provide evidence of pain and suggest the need for analgesia protocols. No previous work has attempted to assess the effects painful procedures on sea lions while they are anaesthetized. Sea lion physiological and behavioural responses at the time of branding were monitored in Chapter 6. Animals thought to be at a surgical plane of anaesthesia exhibited increased heart and breath rates, as well as various behavioural displays, at the time of branding. These results indicate the presence of nociception, possibly even pain, at the time of branding and suggest that isoflurane at the level administered is not sufficient to maintain a surgical plane of anaesthesia in sea lions during the branding procedure (Haskins, 2007).  7.2.1 Future branding research Despite the recent advances in veterinary analgesia and the controversy surrounding hot-iron branding (Dalton, 2005; Beausoleil and Mellor, 2007; McMahon et al., 2007), no research has reported using analgesia during or following branding. Unless there is convincing scientific evidence to supports withholding analgesics, animals that are conscious and experiencing pain should be administered pain medications (Antognini et al., 2005). Future research should be designed to test the efficacy of analgesics in reducing pain responses to branding. If researchers continue to anaesthetize animals during branding, then the administration of peri-operative analgesia, such as a local nerve block or opioid, or the use of orally or intra-muscularly administered analgesics, may help reduce pain responses (Antognini et al., 2005). Additionally, the use of post-operative analgesia such as opioids or  129  local anaesthetic gels and sprays applied after branding (if the animal is not returning directly to the water) may help to reduce pain responses in the days after branding. The human burn literature shows that the pain due to burn injuries lasts until the burn wound is nearly completely healed (Hanafiah et al., 2008). An increased sensitivity to pain, hyperalgesia, from inflammation may be present in the weeks after branding. Prolonged hyperalgesia has been witnessed for up to 28 d in rats that had experienced partial-thickness burns (Summer et al., 2007). These results suggest that the pain associated with the healing of the brands also warrants investigation. Understanding the acute responses, as well as the longer-term effects on the welfare of the animal, will enable researchers to more fully evaluate the effects of hot-iron branding and allow policymakers better evidence to decide whether and when this should be employed as a marking method in the field.  7.3 Balancing animal welfare considerations with research goals The research conducted in this dissertation was conducted in a captive, controlled setting, which may not be truly representative of what would occur if these animals were branded or underwent abdominal surgery in their natural environment. The goal in the application of most technology, such as hot-iron branding and telemetry device attachment or implantation, is to place the marks or devices on ‘wild’ pinnipeds, which would mean there is little to no post-operative care. If the animals are released back into nature when they are still showing behavioural responses to surgery they may be at increased risk of predation. The behavioural effects may also influence foraging abilities or normal activity patterns, which if they persist for an extended period may make the animal more susceptible to disease or injury. Animal researchers must balance their moral responsibility to minimize pain with the  130  risks of using analgesia and anaesthesia both to the animal, the researcher, and possibly the risks to the environment (i.e., are there effects of the drugs on the environment and local ecosystems). Careful consideration of all options available is necessary to minimize possible unwanted side effects from anaesthesia or analgesia. It is suggested that prior to the use of new analgesics or anaesthetics, drug protocols be tested in a controlled, captive setting to account for the possible side effects. In summary, balancing all aspects of the marking procedure including capture, handling, and anaesthesia and analgesia side effects, will help meet the needs of the individual animal’s welfare and the goals of the research program.  7.4 Future directions in pain assessment research To understand the degree of pain experienced would require supplementary techniques. For example, neurophysiological techniques such as the electroencephalograph (EEG) are used in veterinary science to measure the cortical response to complex pain stimulus and to provide information about pain perception (Murrell and Johnson, 2006). Different EEG variables, such as median frequency (F50) and 95% spectral edge frequency (F95), are correlated with distinct aspects of central nervous system function. F95 has been found to be a more reliable and sensitive indicator of anaesthesia than F50 (Johnson and Taylor, 1998; Antognini et al., 2000). F50 is correlated with pain in humans (Chen et al. 1983) and is considered a reliable marker of pain perception in rats, horses, lambs, calves and deer (Otto et al. 1996; Murrell et al. 2003; Johnson et al. 2005a; Johnson et al. 2005b; Gibson et al. 2007; Murrell et al. 2007). The minimal anaesthesia model is a sensitive and objective method developed to assess responses to noxious stimulation of animals under a ‘light’ plane of general anaesthesia by removing the influences of behaviour and physiological responses  131  typically witnessed in conscious animals (Murrell et al. 2003; Johnson et al., 2005a). This model allows researchers to assess changes in EEG frequency to provide information on analgesic and anaesthetic effectiveness. The advantage of EEG technology is that it enables researchers to monitor adequacy and efficacy of anaesthesia and analgesia. However, the disadvantage is that the large amount of data generated can typically only be interpreted by trained personnel, making it difficult to use EEG as a standard monitoring tool. The bispectral index (BIS) is a neurophysiological technique that is used to assess level of consciousness in human patients under general anaesthesia (Murrell and Johnson, 2006; Avidan et al., 2008). Interpretations of BIS do not require extensive knowledge of EEG technology. A BIS value is generated from an algorithmic analysis of a patient's EEG, with the data output given as a single number, ranging from 0 (isoelectric EEG) to 100 (fully conscious). BIS technology has yet to be validated in veterinary science and may provide a valuable tool for use in pain assessment and management (Murrell and Johnson, 2006). Another technique to assess the physiological responses of animals to pain stimuli would involve measurements of heart rate variability (as discussed in Chapter 6). Measurements of heart rate variability would allow for a greater interpretation of autonomic nervous system activity (von Borell et al., 2007). Pain researchers have also tested self-selection of analgesic drugs in animals presented with painful situations. For example, Danbury et al. (2000) allowed lame and sound chickens to choose between different coloured feed with and without the analgesic carprofen. The two feeds were then offered to the chickens at the same time. The lame chickens consumed more analgesic feed than the sound chickens, which improved the lame  132  chickens’ walking ability. Self-selection of analgesia has also been demonstrated in polyarthritic rats (Colpaert et al., 1982) and adjuvant monoarthritic rats (Kupers and Gybels, 1995).  7.5 Conclusions The objectives of this dissertation were to provide a scientific basis for the evaluation of different marking and tagging techniques used in sea lions and to provide information on how to mitigate the potential negative effects. Results from Chapters 3-6 demonstrate that there are welfare concerns associated with abdominal surgery and hot-iron branding of sea lions. In summary, six of the seven behaviours measured in response to abdominal surgery changed, regardless of analgesic treatment (Chapters 3, 4); four of these behaviours still differed at 2 wk after surgery (Chapter 3). These results indicate that neither analgesic protocol was entirely effective at controlling post-operative pain. Four of the six behaviours monitored after hot-iron branding changed in the 3 d after branding (Chapter 5), suggesting the presence of post-operative pain. Sea lions’ immediate responses to hot-iron branding included increased heart and breathing rates, as well as movements (Chapter 6). These results indicate that the sea lions were not at an adequate plane of anaesthesia to block the sympathetic and somatic responses to pain. The presence of intra- and post-operative pain from branding, and the presence of post-operative pain for 2 wk after abdominal surgery, demonstrates the need to investigate more robust pain management protocols. Alternative analgesia protocols should be tried,  133  including the use of multimodal peri-operative analgesia, to help reduce the pain associated with abdominal surgery and branding.  7.6 Personal recommendations Based on the results in this dissertation, I have suggested that different analgesic protocols be thoroughly investigated; however, there are alternatives to this recommendation that could be considered. The first option would be to continue with abdominal surgery and hot-iron branding using the current handling protocols. This may be easiest for wildlife managers, as the costs of testing and implementing additional analgesia will not be incurred; however, as revealed in my dissertation I identified behaviours indicative of pain, which pose a welfare concern. A second option is to implement the use of analgesia without specific protocol testing. This option may also pose a welfare concern, as the animal may experience side effects or no analgesia. A third option is to implement a research protocol in a controlled, captive setting that would allow for proper testing of analgesia. This option would allow researchers to meet the goals of their research program, while taking into account the individual animal welfare concerns associated with the marking procedures. The final option would be to discontinue the use of current methods of abdominal surgery and branding. This would allow for the individual animal welfare concerns to be addressed; however, the reason for implementing procedures such as hot-iron branding is to identify individuals throughout their life and obtain population level data. If this option were implemented, it would counteract this conservation effort. Alternative methods of marking sea lions could be investigated.  134  My personal recommendation is to focus our research on finding effective pain management protocols, which includes testing analgesic side effects. I also support finding alternative permanent marking techniques that could eventually replace hot-iron branding. The animal welfare concerns associated with invasive marking procedures need to be seriously considered when implementing a marking technique in field research, and this needs to be weighed against the conservation research goals. If the pain associated with invasive marking procedures cannot be mitigated, the discontinuation of these procedures and the implementation of alternative, less invasive procedures should be considered.  135  7.7 References Antognini, J., Wang, X.W., Carstens. E. 2000. Isoflurane anaesthetic depth in goats monitored using the bispectral index of the electroencephalogram. Veterinary Research Communications 24: 361–370. Antognini, J., Barter, L., Carstens. E. 2005 Movement as an index of anesthetic depth in humans and experimental animals. Comparative Medicine 55: 413-418. Avidan, M.S., Zhang, L., Burnside, B.A., Finkel, K.J., Searleman, A.C., Selvidge, J.S., Saager, L., Turner, M.S., Rao, S., Bottros, M., Hantler, C., Jacobsohn, E., Evers, A.S. 2008. Anesthesia awareness and the bispectral index. The New England Journal of Medicine 358: 1097-1108. Beausoleil, N. J., Mellor, D.J. 2007. Investigator responsibilities and animal welfare issues raised by hot branding of pinnipeds. Australian Veterinary Journal 85: 484-485. Canadian Council on Animal Care (CCAC). 2003. CCAC guidelines on: The care and use of wildlife. Available at http://www.ccac.ca/en/CCAC_Programs/Guidelines_ Policies/GDLINES/Wildlife/Wildlife.pdf. Chen A.C.N., Dworkin, S.F., Drangholt, M.T. 1983. Cortical power spectral analysis of acute pathophysiological pain. International Journal of Neuroscience 18: 269–278. Colpaert, F.C., Meert, T., DeWitte, P., Schmitt, P. 1982. Further evidence validating adjuvant arthritis as an experimental model of chronic pain in the rat. Life Sciences 31: 67-75. Dalton, R. 2005. Animal-rights group sues over ‘disturbing’ work on sea lions. Nature 436: 315. Danbury, T.C., Weeks, C.A., Chambers, J.P., Waterman-Pearson, A.E., Kestin, S.C. 2000. Self-selection of the analgesic drug carprofen by lame broiler chickens. Veterinary Record 146: 307-311. Daoust, P., Fowler, G.M., Stobo, W.T. 2006. Comparison of the healing process in hot and cold brands applied to harbour seal pups (Phoca vitulina). Wildlife Research 33: 361-372. Dobromylskyj, P., Flecknell, P.A., Lascelles, B.D., Pascoe, P.J., Taylor, P., WatermanPearson, A.. 2001. Management of postoperative and other acute pain. Pages 81-145 in Pain management in animals. Flecknell, P. and A. Waterman-Pearson, (Eds.), Harcourt Publishers Limited, London. Friend, M., Toweill, D.E., Brownell, R.L.,Nettles, V.F., Davis, D.S., Foreyt, W.J.. 1994. Guidelines for proper care and use of wildlife in field research. Pages 96-124 in Research and management techniques for wildlife and habitats. T.A. Bookhour, (Ed.) Wildlife Society, Bethesda, MD. 136  Gibson T.J., Johnson, C.B., Stafford, K.J., Mitchinson S.L., Mellor, D.J. 2007. Validation of the acute electroencephalographic responses of calves to noxious stimulus with scoop dehorning. New Zealand Veterinary Journal 55: 152–157. Hanafiah, Z., Potparic, O., Fernandez, T. 2008. Addressing pain in burn injury. Current Anaesthesia and Critical Care 19: 287-292. Haskins, S.C. 2007. Monitoring anesthetized patients. Pages 533-560 in Lumb and Jones’ Veterinary Anesthesia and Analgesia, 4th Edition. Tranquilli, W.J., Thurmon, J.C. and Grimm, K.A. (Eds.) Blackwell Published, Iowa, USA. Johnson, C.B., Taylor, P.M. 1998. Comparison of the effects of halothane, isoflurane and methoxyflurane on the electroencephalogram of the horses. British Journal of Anaesthesia 81: 748–753. Johnson, C., Wilson, P., Woodbury, M., Caulkett, N. 2005a. Comparison of analgesic techniques for antler removal in halothane-anaesthetized red deer (Cervus elaphus). Veterinary Anaesthesia and Analgesia 32: 61–71. Johnson, C.B., Stafford, K.J., Sylvester, S.P., Ward, R.N., Mitchinson, S., Mellor, D.J. 2005b. Effects of age on the electroencephalographic response to castration in lambs anaesthetised using halothane in oxygen. New Zealand Veterinary Journal 53: 433-437. Kupers, R., Gybels, J. 1995. The consumption of fentanyl is increased in rats with nociceptive but not with neuropathic pain. Pain 60: 137-141. McMahon, C. 2007. Branding the sea branders: what does the research say about seal branding? Australian Veterinary Journal 85: 482-483. McMahon, C.R., Bradshaw, C.J.A., Hays, G.C. 2007. Applying the heat the research techniques for species conservation. Conservation Biology 21: 271-273. McMahon, C.R., Burton, H.R., Vandenhoff, J., Woods, R., Bradshaw C.J.A. 2006. Assessing hot-iron and cryo-branding for permanently marking southern elephant seals. Journal of Wildlife Management 70: 1484-1489. Mellish, J., Calkins, D., Christen, D., Horning, M., Rea, L., Atkinson, S. 2006. Temporary captivity as a research tool: comprehensive study of wild pinnipeds under controlled conditions. Aquatic Mammals 32: 58-65. Mellish, J., Hennen, D., Thomton, J., Petrauskas, L., Atkinson, S., Calkins, D. 2007. Permanent marking in an endangered species: physiological response to hot branding in Steller sea lions (Eumetopias jubatus). Wildlife Research 34: 1-6.  137  Murray, D.L., Fuller, M.R. 2000. A critical review of the effects of marking on the biology of vertebrates. Pages 15-64 in Research techniques in animal ecology. L. Boitani and T.K. Fuller, (Eds.) Columbia University Press, New York, NY. Murrell, J.C., Johnson, C.B. 2006. Neurophysiological techniques to assess pain in animals. Journal of Veterinary Pharmacology and Therapeutics 29: 325-335. Murrell, J., Johnson, C., White, K., Taylor, P., Haberham, Z., Waterman-Pearson, A. 2003. Changes in the EEG during castration in horses and ponies anasethetized with halothane. Veterinary Anaesthesia and Analgesia 30: 138–146. Murrell, J.C., Mitchinson, S. L., Waters, D., Johnson, C.B. 2007. Comparative effect of thermal, mechanical, and electrical noxious stimuli on the electroencephalogram of the rat. British Journal of Anaesthesia 98: 366–371. Nietfeld, M.T., Barrett M.W., Silvy, N. 1994. Wildlife marking techniques. Pages 140-168 in Research and management techniques for wildlife and habitats, Fifth ed. T.A. Bookhout, (Ed.) Wildlife Society, Bethesda, MD. Nolan, A.M. 2000. Pharmacology of analgesic drugs. Pages 21-52 in Pain management in animals. Flecknell, P., and A. Waterman-Pearson (Eds.) Harcourt Publishers Limited, London. Otto, K., Voigt, S., Piepenbrock, S., Deegen, E., Short, C. 1996. Differences in the quantitated electroencephalographic variables during surgical stimulation of horses anaesthetized with isoflurane. Veterinary Anaesthesia and Analgesia 20: 362–371. Ray, J.E., Wade, D.N., Graham, G.G., Day, R.O. 1979. Pharmacokinetics of carprofen in plasma and synovial fluid. Journal of Clinical Pharmacology 19: 635-643 Slingsby, L.S., Waterman-Pearson, A.E. 2001. Analgesic effects in dogs of carprofen and pethidine together compared with the effects of either drug alone. Veterinary Record 148: 441-444. Summer G.J., Dina, O.A., Levine, J.D. 2007. Enhanced inflammatory hyperalgesia after recovery from burn injury. Burns 33:1021–1026. Task Force of the European Society of Cardiology and the North American Society of Pacing and Electrophysiology. 1996. Heart rate variability: standards of measurement, physiological interpretation and clinical use. Circulation 93:1043-1065. van den Hoff, J., Sumner, M.D, Field, I.C., Bradshaw, C.J.A., Burton, H.R., McMahon, C.R. 2004. Temporal changes in the quality of hot-iron brands on elephant seal (Mirounga leonine) pups. Wildlife Research 31: 619-629.  138  Vinuela-Fernandez, I., Jones, E., Welsh, E.M., Fleetwood-Walker, S.M. 2007. Pain mechanisms and their implication for the management of pain in farm and companion animals. The Veterinary Journal 174: 227-239. von Borell, E., Langbein, J., Depres, G., Hansen, S., Leterrier, C., Marchant-Forde, J., Marchant-Forde, R., Minero, M., Mohr, E., Prunier, A.,Valance, D., Veisser, I. 2007. Heart rate variability as a measure of autonomic regulation of cardiac activity for assessing stress and welfare in farm animals – A review. Physiology & Behaviour 92: 293-316.  139  Appendix I – UBC Animal Care Certificates  140  141  

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.24.1-0071136/manifest

Comment

Related Items