INDICATORS OF INDIVIDUAL AND POPULATION HEALTH IN THE VANCOUVER ISLAND MARMOT (MARMOTA VANCOUVERENSIS) BY MALCOLM LEE MCADIE D.V.M., Western College of Veterinary Medicine, 1987 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN ENVIRONMENTAL SCIENCES Thompson Rivers University Kamloops, British Columbia, Canada November 2018 Thesis examining committee: Karl Larsen (PhD), Thesis Supervisor, Natural Resource Sciences, Thompson Rivers University Craig Stephen (DVM, PhD), Thesis Supervisor, Executive Director, Canadian Cooperative Wildlife Health Cooperative David Hill (PhD), Committee Member, Faculty of Arts, Thompson Rivers University Todd Shury (DVM, PhD), External Examiner, Wildlife Health Specialist, Parks Canada ii Thesis Supervisors: Dr. Karl Larsen and Dr. Craig Stephen ABSTRACT The Vancouver Island Marmot (Marmota vancouverensis) is an endangered rodent endemic to the mountains of Vancouver Island, British Columbia, Canada. Following population declines in the 1980s and 1990s, an intensive captive breeding and reintroduction program was initiated involving three Canadian zoos and a purpose-built, subalpine facility on Vancouver Island. From 1997 to 2017, 660 marmots were associated with the captive program, including 63 wild-born individuals captured for breeding and 597 marmots born and weaned in captivity. Reintroductions began in 2003 and by 2017 a total of 501 marmots had been released. Although this significantly increased the wild population from its low point in 2003, conservation of the Vancouver Island Marmot (VIM) continues to involve intensive ex situ management, reintroductions, and translocations. Health and disease surveillence is fundamental to the success of conservation programs like the marmot recovery project. This thesis builds upon our understanding of VIM health by describing and evaluating select health parameters, to determine baseline characteristics for VIM under its different treatments (wild, captive and captive-release) and to identify potential risk factors that may influence the health of the marmot’s population and its capacity to achieve recovery objectives. The analysis involved data that was collected between 1992 and 2016, and included 1,106 VIM blood profiles, 3,174 physical examinations, 140 post mortem examinations and 533 field mortality records. VIM hematology and serum biochemistry reference ranges were calculated as a baseline metric and were qualitatively comparable to published values for other iii rodent species. Leukogram and protein values were found to have potential utility as a quantitative measure for comparing VIM management groups. There were significant differences in the clinical and pathological data collected from captive and free-ranging (captive-release and wild) marmots. Captives could be monitored with greater intensity and to an older age, due to increased longevity. A host of clinical and pathological disorders were described in captive marmots, including age-related, management-related, and congenital problems. There was a paucity of health conditions identified in free-ranging VIM and this could be due to a fundamental lack of disease, or limited opportunities to conduct post mortem examinations or evaluate compromised individuals in the field. The analysis did not identify any specific infectious agents that represented a generalized population threat to VIM. Cardiomyopathy and neoplasia, which occurred in older individuals, were the most consequential health complications for captive marmots. Implantation of abdominal radio-transmitters was not found to impact marmot health and was important for identifying mortalities in the field. The first wild hibernation represented a time of significant mortality for VIM released from captivity. However, reintroduced marmots that survived their first year in the wild were comparable to their wild-born counterparts with respect to hibernation success and clinical presentation. Predators continued to represent a major cause of mortality for free-ranging marmots. In the absence of other identified health threats, predation and reduced hibernation success of captive-release marmots appear to be significant factors limiting the health and potential recovery of the in situ population. Keywords: marmot, endangered, recovery, health, disease, surveillance iv TABLE OF CONTENTS ABSTRACT ................................................................................................................................ ii TABLE OF CONTENTS ............................................................................................................. iv ACKNOWLEDGEMENTS ........................................................................................................ vii DEDICATION ........................................................................................................................... ix LIST OF FIGURES ...................................................................................................................... x LIST OF TABLES ...................................................................................................................... xii CHAPTER 1: THESIS INTRODUCTION ......................................................................................1 Health Risks in Threatened Species .........................................................................2 Individual Health ........................................................................................................4 Population Health .......................................................................................................4 The Vancouver Island Marmot .................................................................................6 Marmot Recovery and Current Status ...................................................................10 Thesis Structure .........................................................................................................14 Literature Cited .........................................................................................................15 CHAPTER 2: HEMATOLOGICAL AND SERUM BIOCHEMICAL PARAMETERS IN THE VANCOUVER ISLAND MARMOT: REFERENCE RANGES AND A COMPARISON OF VALUES BETWEEN MANAGEMENT GROUPS Introduction ...............................................................................................................20 Materials and Methods.............................................................................................22 Results .........................................................................................................................29 Discussion ..................................................................................................................56 Literature Cited .........................................................................................................60 v CHAPTER 3: SURVEILLANCE OF VANCOUVER ISLAND MARMOT HEALTH USING CLINICAL AND POST MORTEM EXAMINATIONS, AND FIELD MORTALITY DETERMINATION Introduction ...............................................................................................................64 Materials and Methods.............................................................................................67 Results .........................................................................................................................78 Discussion ..................................................................................................................98 Literature Cited .......................................................................................................104 CHAPTER 4: CONCLUSION ...................................................................................................107 Literature Cited .......................................................................................................116 vi LIST OF APPENDICES APPENDIX A. Vancouver Island Marmot captive population numbers, 1997 to 2018 ..... ....................................................................................................................................118 APPENDIX B. Descriptive statistics for hematological variables from clinically normal or healthy Vancouver Island Marmots before & after removal of outliers ....119 APPENDIX C. Descriptive statistics for serological variables from clinically normal or healthy Vancouver Island Marmots before & after removal of outliers .........121 APPENDIX D. Characteristics of implantable abdominal transmitters used in Vancouver Island Marmots 1992 to 2018 .............................................................124 APPENDIX E. Summary of Vancouver Island Marmot implant surgeries, 1992 to 2018 ....................................................................................................................................126 APPENDIX F. Trapping, handling, anesthesia & implant surgery techniques in Vancouver Island Marmots ...................................................................................127 APPENDIX G. Summary of morbidity & mortality in captive & free-ranging Vancouver Island Marmots 1992 to 2016 .............................................................136 APPENDIX H. Summary of annual mortalities in free-ranging Vancouver Island Marmots 1992 to 2016 .............................................................................................138 APPENDIX I. A comparison of common body condition indices in wild & captive male Vancouver Island Marmots..........................................................................139 vii ACKNOWLEDGEMENTS Dave Fraser, the Endangered Species Specialist for British Columbia’s Ministry of Environment (and a former member of the Vancouver Island Marmot Recovery Team), once told me that the most successful conservation efforts were the ones driven by individuals who are passionate about the cause. Over many years, marmot recovery, and my own humble thesis, have benefited greatly from the hard work, expertise, and passion of many people. This includes the animal care, curatorial, and veterinary staffs at the Toronto Zoo and the Calgary Zoo, and Gordon Blankstein and the workforce at the Mountain View Conservation and Breeding Society. Collette Howell, Rick Wenman and Debbie Rempel were instrumental in developing the initial protocols for marmot husbandry and breeding. Dr. Doug Whiteside, Dr. Sandie Black, Dr. Graham Crawshaw, Dr. Chris Dutton and many other veterinarians, have made important contributions to marmot health. The staff at the Tony Barrett Mount Washington Marmot Recovery Centre, particularly Louise Dykslag and Alana Buchanan, contributed many years of diligent animal care. A number of pathologists at the University of Guelph, the Calgary Zoo and the Animal Health Centre (AHC) in Abbotsford conducted marmot post mortems. Dr. Stephen Raverty (AHC) deserves special recognition for his thoroughness, expertise and the number of marmot cases he examined. He went out of his way on many occasions to provide us with his insights and support. The Vancouver Island Marmot Recovery Team and the Captive Management Group grappled with difficult decisions and dealt with contentious issues in a consistently thoughtful and respectful manner. Tremendous leadership was shown by the Recovery Team Chairs - Doug Janz, Don Doyle and Sean Pendergast (current) viii - and by the Executive Directors of the Marmot Recovery Foundation (MRF), the late Tony Barrett, the late Robert Huber, Viki Jackson and Adam Taylor (current). The late Jim Walker, former Chair of the MRF Board, and many MRF board members, were vital advocates for the marmots. An army of field personnel endured extreme temperatures, biting insects, lumpy ground, challenging terrain, predators and many long days and nights. This included Cheyney Jackson (current MRF Field Coordinator), Mike Lester, Jerry MacDermott, Sean Pendergast, Crystal Reid, Chris White and many, many others. Dr. Andrew Bryant was important to VIM and the project in many ways, and along with Tony Barrett and Stan Coleman, was instrumental in getting the MRF off the ground. Dr. Ken Langelier developed many of the original techniques used for surgically implanting radio-transmitters. Robin Campbell and the North Island Wildlife Recovery Association provided support in a multitude of ways. John Carnio was instrumental in starting the captive breeding program at the Toronto Zoo and has acted as the VIM Studbook Keeper since its inception. His sage wisdom has been valued for many years. His successor at the Toronto Zoo, Maria Franke, has been a strong voice for the marmots. Dr. Helen Schwantje has been an instrumental and multi-faceted contributor and promoter of marmot health. Dr. David Hill (a member of my thesis committee) spent valuable time helping me learn the basics of statistics, and Dr. Todd Shury was a welcome addition as my external examiner. I would like to thank my supervisors, Dr. Craig Stephen and Dr. Karl Larsen, for their friendship, guidance and patience. It was very much appreciated. And a thanks to the VIM themselves, who live their lives as marmots, and do not stress about being critically endangered. A lesson to us all. ix DEDICATION This thesis is dedicated to Marnie, Steven, Greg, Laura, Vern and Margaret. You didn’t always understand my passion for wildlife, but you accepted and supported it nevertheless. Haida (right) was born in 2002 at the Mountain View Conservation and Breeding Society in Langley, British Columbia and became the first captive-release female to breed in the wild. Her descendants continue to survive at the Haley Lake Ecological Reserve on Vancouver Island. Photo: Oli Gardner People often ask me ‘What are marmots good for?’, I ask them “What are you good for?” Dr. Kenneth B. Armitage x LIST OF FIGURES Figure 1.1. Historical distribution of the Vancouver Island Marmot ..............................8 Figure 1.2. Size of the Vancouver Island Marmot population from 1997 to 2017 ......13 Figure 2.1. Schematic diagram illustrating the relationships and sample size of the Vancouver Island Marmot blood sampling categories .......................................26 Figure 2.2. Annual distribution of 1,106 blood samples collected from Vancouver Island Marmots 1992 to 2015 ...................................................................................31 Figure 2.3. Monthly distribution of 1,106 blood samples collected from captive and free-ranging Vancouver Island Marmots ..............................................................31 Figure 2.4. Sex and age distribution of 1,106 blood samples collected from captive and free-ranging Vancouver Island Marmots ......................................................32 Figure 2.5. Comparison of age distribution of 1,106 blood samples collected from captive and free-ranging Vancouver Island Marmots.........................................32 Figure 2.6. Changes in total white blood cells and total protein from April to October for clinically normal Vancouver Island Marmots ................................................42 Figure 2.7. Boxplot comparisons of red blood cells and hemoglobin between June and October for clinically normal Vancouver Island Marmots .........................43 Figure 2.8. Change in red blood cells and hemoglobin for clinically normal Vancouver Island Marmots according to age .......................................................44 Figure 2.9. Change in glucose and amylase for clinically normal Vancouver Island Marmots according to age......................................................................................................... 45 Figure 2.10. Boxplots comparing male and female values for hematocrit and amylase in Vancouver Island Marmots .................................................................................46 xi Figure 3.1. Age comparison of physical examinations conducted on wild and captive Vancouver Island Marmots .....................................................................................81 Figure 3.2. Sex-age distribution of 109 mortalities in captive, weaned Vancouver Island Marmots 1997 to 2016 ...................................................................................88 Figure 3.3. Sex-age distribution of 518 mortalities in free-ranging Vancouver Island Marmots 1992 to 2016 ...............................................................................................88 Figure 3.4. Yearly predation mortalities of free-ranging Vancouver Island Marmots 1992 to 2016 ................................................................................................................96 xii LIST OF TABLES Table 2.1. Reference intervals for 16 hematological parameters from clinically healthy Vancouver Island Marmots .......................................................................33 Table 2.2. Reference intervals for 30 serum biochemistry parameters from clinically healthy Vancouver Island Marmots .......................................................................34 Table 2.3. Vancouver Island Marmot hematological and serological mean values compared to published means for the Woodchuck (Marmota monax) and normal values published for Mice (Mus musculus), Rats (Rattus norvegicus), Gerbils (Meriones unguiculatus), and Guinea Pigs (Cavia porcellus) ....................36 Table 2.4. Hematology values comparing healthy captive (un-implanted), wild and captive-release Vancouver Island Marmots ..........................................................47 Table 2.5. Serum biochemistry values comparing healthy, captive (un-implanted), wild and captive-release Vancouver Island marmots .........................................48 Table 2.6. Hematology values comparing (i) clinically normal, non-implanted, (ii) clinically normal, implanted, and (iii) clinically abnormal Vancouver Island Marmots .....................................................................................................................50 Table 2.7. Serum biochemistry values comparing clinically normal non-implanted and implanted and clinically abnormal Vancouver Island Marmots ...............51 Table 2.8. Summary of statistical and clinical significance in hematology parameters between specific treatments or groups of Vancouver Island Marmots ............53 Table 2.9. Summary of statistical and clinical significance in serology parameters between specific treatments or groups of Vancouver Island Marmots ............54 Table 3.1. Circumstances of 3,174 physical examinations conducted on captive and free-ranging Vancouver Island Marmots ..............................................................80 Table 3.2. Clinical conditions initially identified from 3,174 Vancouver Island Marmot physical examinations ...............................................................................85 xiii Table 3.3. Categories of mortality observed in 109 captive Vancouver Island Marmots 1997 to 2016 ...............................................................................................87 Table 3.4. Anatomical description of neoplasia in 26 captive Vancouver Island Marmots 1997 to 2016 ...............................................................................................89 Table 3.5. Causes of mortality for wild, translocated, captive-release and preconditioned Vancouver Island Marmots ...............................................................94 Table 3.6. Predator mortality for wild, translocated, captive-release and preconditioned Vancouver Island Marmots ...............................................................95 Table 3.7. Post mortem diagnosis in 8 (7 telemetered, 1 non-telemetered) wild Vancouver Island Marmots 1992 – 2016 ................................................................97 Table 3.8 Post mortem diagnosis in 23 captive-release Vancouver Island Marmots 2003 – 2016..................................................................................................................97 1 CHAPTER 1 THESIS INTRODUCTION To prevent extinction and facilitate recovery, many conservation programs have undertaken intensive management of endangered species. In the case of the critically endangered Vancouver Island Marmot (Marmota vancouverensis) this has involved captive breeding and conservation translocations, the latter defined as the deliberate movement of individuals from one location, either captive or wild, to free release in another location, to restore extirpated populations or to support existing small populations (Convention on Biological Diversity, 2013; Ewan et al. 2012; Williams & Hoffman, 2009; Teixeira, et al. 2007; Rout et al., 2007; IUCN, 2002; Fischer & Lindenmayer, 2000; Ebenhard, 1995). Dwindling numbers and artificial manipulation, including animal movements and ex situ management, have the potential to negatively impact the health of a threatened species (Daszak, et al. 2001; Snyder, et al. 1996). Although comprehensive tracking of health and health effects in imperiled wildlife populations is essential for their successful long-term maintenance and recovery, and for the timely recognition and mitigation of potential threats, effective health monitoring and its implications represents a significant challenge for many conservation programs (Delahay et al. 2009). The objective of this thesis is to build upon our current understanding of Vancouver Island Marmot (VIM) health by describing and evaluating select health parameters, to determine what is expected for this species under different treatments (wild, captive and captive-release) and to identify possible risk factors associated with small population size and conservation management that may be 2 influencing the health of the marmot’s population and its relative capacity to achieve defined recovery objectives. HEALTH RISKS IN THREATENED SPECIES Declines in population size can result in a loss of genetic diversity that may compromise an endangered species’ capacity to cope with current conditions or adapt to future change (Jamieson, 2010). Decreased heterozygosity can have multiple consequences including a reduction in rigour, growth, fecundity, survival, and immunocompetence, and it may also lead to the accumulation and expression of deleterious alleles (Ewan et al. 2012; Williams and Hoffman, 2009). Captivity can significantly alter animals (Darwin, 1859) and artificial conditions may promote the occurrence of heritable or acquired traits that reduce the fitness of individuals being returned to the wild (Kreger et al. 2005). Many translocation programs using captivebred animals have been less successful than those ultilizing wild-born animals (Robert, 2009; Teixeira et al. 2007, Jule, Leavor, & Lea, 2008; Fischer & Lindenmayer, 2000). The post-release success of captive animals may be limited by physical, physiological and behavorial deficiencies, including inadequate physical conditioning, underdeveloped locomotor abilities, morphological changes, alteration of reproductive and metabollic cycles, increased stress, inadequate or inappropriate anti-predator or threat responses, overt boldness or aggression, increased docility, alterred sociocognitive abilities, and an inability to recognize or procure appropriate foods (Bonato et al. 2013; Teixeira et al. 2007; Hare, et al., 2005; McPhee, 2003; Concannon et al. 1997). Rapid genetic changes associated with domestication may occur within a single generation in certain taxa (De Mestral & Herbinger, 2013) and 3 some abberant characteristics may become more pronounced or more variable following multiple generations in captivity (McPhee, 2003). Captive confinement also can result in the artificial intensification of endemic parasites, or alternatively it may result in a reduction of acquired resistence due to the disruption of natural relationships that are normally maintained between a host and its commensal pathogens and parasites (Mathews et al. 2005). Most zoological institutions maintain geographically-varied collections of animals from a diversity of sources, each with their own spectrum of infectious agents (Snyder, et al. 1996). In addition, captive facilities may inadvertently harbour pest species, which provide an additional source for the introduction of exotic pathogens. Even with the safeguard of good biosecurity, there is always the potential risk that an ex situ recovery population maintained within a zoo will be exposed to a novel pathogen. This potential risk cannot be fully quantified because it is not realistically possible to determine the full spectrum of suseptibilty that an endangered species has to novel infectious agents. Whenever animals are translocated from one environment to another (be it wild to captive, captive to captive, captive to wild or wild to wild) there is a risk to the health of the individuals being moved and a risk to the health of extant individuals or populations (Leighton, 2002; Woodford, 1993). Translocated animals may serve as vectors for the introduction of novel pathogens to naïve recipient populations, or alternatively, they may be naïve to the transmissible agents that exist within the extant conspecific or sympatric populations at their release site. 4 INDIVIDUAL HEALTH To survive and propagate, organisms must co-ordinate complex assemblages of functional and metabolic characteristics that allow for the maintenance of intrinsic equilibrium, or homeostasis. To be effective, this physiological balance must be maintained within the typical range of ambient conditions to which a species has become adapted, and to some extent, it must also be preserved in response to atypical perturbations (Ryser-Degiorgis, 2013). Within a species, there is individual variability in the capacity to cope or self-manage. Those individuals that are less competent at regulating biological integrity or those that are less able to maintain function during times of challenge or stress, have diminished resilience and are potentially inferior with respect to growth, reproduction, and survival (Darwin, 1859). An individual’s capacity to maintain homeostasis, well-being, and normal life functions, can be equated to its comparative state of health. A reduced ability to support homeostasis can result in biological impairment and this can be associated with imbalance or disease (Ryser-Degiorgis, 2013). If health is used in the context of a continuum or relative state (i.e. an individual’s ability to sustain biological processes and its productivity at specific points in life, or cumulatively over a lifetime, compared to conspecifics) it does not merely represent the absence of observable dysfunction or disease (Gunnarsson, 2006). POPULATION HEALTH Although health in individuals can be quantified by measurable characteristics or outcomes relative to those of conspecifics or even individuals of other species, it is much more difficult to define and evaluate the health of populations, species, and the intricate ecosystems of which they are a part (Deem et 5 al. 2008). Many wildlife studies evaluating health have emphasized the description of dysfunction, pathogens, and mortality in populations (Ryser-Degiorgis, 2013). Although the occurrence of infectious and non-infectious disease and the factors that predispose populations to disease are important to comprehensive monitoring and conservation, this type of data represents only one component in the multifaceted context of population health. Health surveillance also can involve the evaluation of many other parameters and condition indices, including growth, physical characteristics and body condition, fecundity, longevity, survival rates, recruitment rates, commensal microflora, behavior, demographics, physiological and immunological measures, and genetic diversity (Robert & Schwanz, 2013; Sackett et al. 2013; Brashares et al. 2010; Mellish et al. 2010; Villers et al. 2008; Mathews et al. 2006; Stevenson & Woods, Jr., 2006; Ostermann et a., 2001). A workable health construct for a species can be based on two basic components - a normative component and a descriptive component (Hanisch, et al. 2012). The normative component of population health seeks to address what should be, by identifying an ideal standard or holistic condition of population well-being, and by determining what is required to restore or maintain that condition (Hanisch, et al. 2012). The ideal, healthy wildlife population could be one that is self– sustaining, and able to survive within its natural habitat, while maintaining its intricate ecological relationships without the need for artificial support or intervention. Such a population should have the capacity to persist for a prolonged, but indeterminate period and avoid premature extinction. It must have the intrinsic ability to cope with existing and future stressors, and demonstrate resiliency and sustainability. Threatened populations, by their very definition, lack the capacity to autonomously achieve these normative ideals of integrity and health. From a 6 conservation and management perspective, the comparative state of fitness or health of a threatened or recovering wildlife population could be its relative ability to achieve and maintain discrete, biologically feasible, recovery objectives. Recovery involves the identification and mitigation of threats to increase the likelihood that a threatened species will continue to persist in the wild (Vancouver Island Marmot Recovery Team, 2008). To achieve recovery there is a need for appropriate intervention and this needs to be supported by social and political will, adequate resources, and technical wherewithal. Therefore, an endangered species’ current and future health also is dependent upon the presence or absence of tangible remedial actions and upon the logistical feasibility of implementing them. The descriptive component of health incorporates the empirical, bio-statistical elements of health and health surveillance (Hanisch et al. 2012). This includes the establishment of baseline parameters to delineate what is normal or expected for a species, how these measures compare between populations exposed to different conditions, and how they reflect on the wellness and population viability of the species in question (Vitali et al, 2011). These parameters should be monitored over time to identify how they change in response to existing conditions (which may be indicative of health deterioration and reduced coping capacity) or to new stresses or challenges. THE VANCOUVER ISLAND MARMOT As both its common and scientific names suggest, the Vancouver Island Marmot (VIM) is a species that is endemic to the insular mountains of Vancouver Island (Swarth, 1912; Swarth, 1911). This large, fossorial sciurid naturally inhabits small, moderately to steeply sloped, south to west facing subalpine meadows 7 between 1000 and 1450 metres elevation (Bryant and Janz 1996). Within recent historical times (i.e. within the last century) its range has extended from several mountains to the immediate north of Lake Cowichan (Latitude 48.94 N, Longitude 124.16 W) on south-central Vancouver Island to Mount Schoen (Latitude 50.16 N, Longitude 126.23 W) which lies approximately 200 kilometers to the northwest (Janz et al. 2000) (Figure 1.1). Due to the low numbers of VIM, limited available natural habitat, restricted geographical range, and endemic status, the species was first listed as endangered in 1978 by the Committee on the Status of Endangered Wildlife in Canada (COSEWIC 1979). Currently, the marmot is listed as critically endangered by the International Union for the Conservation of Nature’s (IUCN) Red List. It also receives legal protection under the federal Species at Risk Act (SARA), the British Columbia Wildlife Act, and the United States Endangered Species Act (Vancouver Island Marmot Recovery Team, 2008). Field inventory conducted in the early to mid-1980s, concentrating primarily in the Nanaimo Lakes region (identified as the “core” area for marmots - which encompasses mountains to the north and north-west of Lake Cowichan and to the east and north-east of the Alberni Inlet) determined that there were approximately 300 to 350 marmots in existence and that the population was stable or increasing. However, beginning in the late 1980s and throughout the 1990s, VIM numbers demonstrated precipitous and progressive declines (Bryant & Page, 2005). By 1998 the wild population had been reduced to less than 100 individuals and by 2003 it was less than 30 (Jackson et al. 2015; Janz et al. 2000). 8 Figure 1.1. Historical distribution of the Vancouver Island Marmot (Marmota vancouverensis), from the time it was first scientifically collected by Harry Swarth in 1910 up to the early 2000s, prior to intensive reintroduction efforts which began in 2003. The elipses delineate the two geographically distinct metapopulations that have been delineated for species recovery; (1) Nanaimo Lakes and (2) Strathcona Park / Mount Washington. Two other sites, (3) Mount Cain and (4) Steamboat Mountain on the Clayoquot Plateau are locations of extralimital, assisted colonization. Map prepared by A.A. Bryant and adapted from the 2000 Update of the National Recovery Plan for the V.I. Marmot KEY Active colonies Inactive sites Solitary marmots 3 Extralimital 2 23 4 1 1 8 9 In the early 1980s it was first determined that a significant portion of the marmot population had recently become established in higher tracts of logged habitat (at elevations above 700 meters) that in the early stages of seral succession, mimicked the characteristics of the marmots’ natural subalpine meadows. However, rapid regeneration of conifers and alders at these sites altered the formerly meadowlike habitat, making them unsuitable for the colonizing marmots. Increased tree cover may also have served to increase the stalking advantages for Cougars (Puma concolor). The increased open habitat created by logging may have facilitated an increase in Golden Eagle (Aquila chrysaetos) numbers and extensive logging roads may have acted as travel corridors for Grey Wolves (Canis lupus), thereby increasing their access and contact with marmots who commonly used these same roads as burrowing substrate. Increased vulnerability to predation from these naturallyoccurring predators may have resulted in marmot declines at these anthropogenic, ephemeral sites (Aaltonen et al. 2009). This resulted in a typical pattern of cut-block colonization and population growth, followed by colony attrition, collapse and eventual extirpation (Bryant, 1996). Most colonies in logged habitat became established in close proximity to natural colonies, which started to exhibit parallel declines, and in many cases, extirpation. It is possible that the logged areas disrupted colony connectivity, genetic exchange and “rescue” of natural colonies by intercepting dispersing marmots, thus interfering with the natural mechanisms that had traditionally perpetuated the marmots’ meta-population (Bryant, 1998). Altered predator-prey relationships on Vancouver Island, including declines in Black-tailed Deer (Odocoileus hemionus), may have also led to increased predation pressure that affected marmots in both natural and logged habitat (Bryant and Page, 2005). 10 Limited historical data exists for the populations in Strathcona Park and Forbidden Plateau, which lie to the northwest of the Alberni Inlet. It is believed that these populations declined over the last few decades, leaving only a single extant colony inhabiting the artificially managed ski-runs at Mount Washington (Bryant, 1998). These northern sites were not under the same development pressures as the southern “core” and the proximate causes of marmot declines in these areas and in the Beaufort Range, which connected the north-west and south-east populations, have not been well established. There is compelling paleontological and archaeological evidence to suggest that this species was once more widespread on Vancouver Island and that its subsequent range contraction was not entirely related to modern anthropogenic influences (Nagorsen et al. 1996). MARMOT RECOVERY AND CURRENT STATUS Based upon extremely low numbers and ongoing population declines, the 2000 National Recovery Plan concluded that although sufficient natural habitat remained, recovery efforts involving only in situ management were unlikely to save the species from extinction. This document stated that “few animals exist for reintroductions or other management activities” and that “it is unlikely that wild populations will suddenly rebound of their own accord (and therefore) Captive breeding and reintroduction present the only chance of increasing populations within a reasonable period of time and minimizing the risk of extinction”. As a result of these determinations, an intensive captive breeding program was initiated for this species in 1997, with the intention of (i) establishing a safeguard against potential catastrophic or stochastic events in the wild, (ii) acting as a long-term genetic reservoir, (iii) determining appropriate management and 11 husbandry techniques for the successful captive maintenance and propagation of Vancouver Island Marmots, (iv) conducting directed research, and (v) providing sufficient numbers of individuals for release and eventual restoration of the wild population (Janz et al. 2000). Since its inception, this ex situ program has involved the participation of three zoological institutions, the Toronto Zoo (TZ), the Calgary Zoo (CZ), and the Mountain View Conservation and Breeding Society in Langley, British Columbia (MVF) along with the construction of a dedicated $1.2 million marmot facility at Mount Washington on Vancouver Island, the Tony Barrett Mount Washington Marmot Recovery Centre (MRC). The VIM captive breeding program reached its numerical peak in 2008 with 177 marmots and 46 breeding pairs. From 2009 to 2015 the program was intentionally down-sized due to diminishing resources and some early reintroduction success (Jackson et al. 2015). Subsequent declines in the wild population following 2012 have prompted a recent restoration of the captive program. As of December 2017, the captive population consists of 49 surviving marmots, including 12 breeding pairs. Overall, there has been a total of 660 individual marmots maintained in captivity, including 63 marmots originally captured from the wild and 597 captive-born marmots. One hundred and twelve mortalities have occurred in captivity and a total of 501 marmots (8 wild-born, 493 captive-born) have been released back to the wild (Vancouver Island Marmot Captive Management Group, 2017). Releases include 167 captive individuals into the Nanaimo Lakes area, 187 into Strathcona Park, 109 to Mount Washington, 16 into the Clayoquot Plateau Provincial Park, and 22 into the Mount Cain / Mount Schoen area. Based upon historical occurrence records, the estimated carrying capacity of suitable habitat patches, and population simulation models, recovery for the 12 Vancouver Island marmot was initially defined as a self-sustaining wild population of 400 to 600 marmots, distributed in three geographically separate metapopulations on Vancouver Island (Vancouver Island Marmot Recovery Team, 2008; Janz, et al. 2000). The potential existence of three independent meta-populations, identified as (i) the Nanaimo Lakes region, (ii) western Strathcona Park and (iii) Mount Washington / Forbidden Plateau, would likely serve to reduce the population’s vulnerability to stochastic or localized threats, and were delineated by the presence of large water bodies, primarily the Alberni Inlet and Buttle Lake, that would act as barriers to dispersing marmots. Due to the potential for habitat connectivity recently identified along the southern end of Buttle Lake, the western Strathcona Park and Mount Washington / Forbidden Plateau were deemed to be a single recovery population in the 2017 Recovery Strategy. Based upon the limitations imposed by a discrete numerical recovery goal (i.e. one without a temporal component), this strategy also re-defined marmot recovery as both geographically distinct meta-populations having a greater than 90% probability of persisting for over 100 years, without augmentation from the captive program (Vancouver Island Marmot Recovery Team, 2017; Jackson et al. 2015). Although there has been some population restoration from its low point in 2003, the VIM continues to be managed by an intensive program involving captive breeding, reintroductions, and translocations. At the end of the 2017 field season population estimates were 70 to 80 individuals in the Nanaimo Lakes metapopulation, 70 to 80 in the Strathcona Park meta-population (including Mount Washington), and an unknown number surviving within Clayoquot Plateau Provincial Park, and at Mount Seth in Schoen Lake Provincial Park. The total wild population is currently estimated to be approximately 150 individuals (Vancouver Island Marmot Recovery Team, November 2017). 13 Figure 1.2. Annual total of individuals in the Vancouver Island Marmot (Marmota vancouverenesis) population from 1997 to 2017. Number of marmots 600 500 Captive Wild captures 400 Releases Wild (estimated) 300 200 100 0 97 99 01 03 05 07 09 11 13 15 17 Year 13 14 THESIS STRUCTURE For the foreseeable future, the critically endangered VIM will be managed by an intensive program of captive breeding and conservation translocations. Low numbers and artificial manipulation pose potential risks that could negatively impact individual and population health and could prevent this species from achieving recovery objectives. Effective health monitoring and a better understanding of normality or baseline characteristics, and identification of possible threats, is essential for the successful long-term conservation of this species. This recovery program has gathered a great range of health information from the marmots’ wild, captive, and captive-release populations. Without further collation of these disparate data, their usefulness for future research, learning, comparison, evidence-based decisions, and management will not be fully realized. In this thesis I compile and analyse these data by describing and evaluating select health parameters. Individual condition indices (hematology and serology, chapter two), clinical assessments (physical examinations, chapter three), and health outcomes (morbidity and mortality, chapter three) are used to establish and compare baseline health characteristics for the VIM under different treatments (wild, captive and captive-release) and to identify possible health threats. This information can be used in the future to recognize changes in VIM health and to guide management decisions. 15 LITERATURE CITED Aaltonen, K., Bryant, A. A., Hostetler, J. A., & Oli, M. K. (2009). Reintroducing endangered Vancouver Island marmots: survival and cause-specific mortality rates of captive-born versus wild-born individuals. Biological Conservation 142, 2181-2190. Bonato, M., Malecki, I. A., Wang, M. D., & Cloete, S. W. (2013). Extensive human presence at an early age of ostriches improves the docility of birds at a later stage of life. Applied Animal Behaviour Science 148, 232-239. Brashares, J. S., Werner, J. R., & Sinclair, A. (2010 ). Social 'meltdown' in the demise of an island endemic: Allee effects and the Vancouver Island marmot. Journal of Animal Ecology 79, 965-973. Bryant, A. A. (1996). Reproduction and persistence of Vancouver Island marmots (Marmota vancouverensis) in natural and logged habitats. Canadian Journal of Zoology, 678-687. Bryant, A. A. (1998). Metapopulation ecology of Vancouver Island marmots (Marmota vancouverensis), PhD thesis. Victoria, B.C.: University of Victoria. Bryant, A. A., & Janz, D. W. (1996). Distribution and abundance of Vancouver Island marmots (Marmota vancouverenisis). Canadian Journal of Zoology (74), 667-677. Bryant, A. A., & Page, R. E. (2005). Timing and causes of mortality in the endangered Vancouver Island marmot (Marmota vancouverensis). Canadian Journal of Zoology (83), 674-682. Concannon, P., Roberts, P., Baldwin, B., & Tennant, B. (1997). 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Journal of Fish Biology 83 (5), 1268-1286. Deem, S. L., Parker, P. G., & Miller, R. E. (2008). Building bridges: connecting the health and conservation professions. The Journal of Tropical Biology and Conservation, 662-665. Delahay, R. J. et al. (eds). (2009). Management of Disease in Wild Mammals. New York: Springer. Ebenhard, T. (1995). Conservation breeding as a tool for saving species from extinction. Tree 10 (11), 438-443. Ewan, J. C., Armstrong, D. P., Parker, P. A., & Seddon, P. J. (2012). In Reintroduction Biology: Integrating Science and Management. Wiley- Blackwell. Fischer, J., & Lindenmayer, D. B. (2000). As assessment of the published results of animal relocations. Biological Conservation 96, 1-11. Gunnarsson, S. (2006). The conceptualisation of health and disease in veterinary medicine. Acta Veterinaria Scandinavica 48 (20). Hanisch, S. L., Riley, S. J., & Nelson, M. P. (2012). Promoting wildlife health or fighting disease: insights from history, philosophy, and science. Wildlife Society Bulletin 36 (3), 477-482. Hare, B., Plyusnina, I., Ignacido, N., Schepina, A., Wrangham, R., & Trut, L. (2005). Social cognitive evolution in captive foxes is a correlated by-product of experimental domestication. Current Biology (15), 226-230. IUCN Technical Guidelines on the Management of Ex-situ Populations for Conservation. (2002). Retrieved from ICUN: http://www.iucn.org/about/work/programmes/species/publications/iucn_gui delines_and__policy__statements/ 17 Jackson, C.; Baker, A.; Doyle, D.; Franke, M.; Jackson, V.; Lloyd, N.; McAdie, M.; Stephens, T.; Traylor-Holzer, K.;. (2015). Vancouver Island Marmot Population and Habitat Viability Assessment Workshop Final Report. Apple Valley, MN: IUCN SSC Conservation Breeding Specialist Group. Jamieson, I. G. (2010). Founder effects, inbreeding, and loss of genetic diversity in four avian reintroduction programs. Conservation Biology 25 (1), 115-123. Janz, D. W., Bryant, A. A., Dawe, N. K., Schwantje, H., Harper, B., Nagorsen, D., . . . Simmons, R. (2000). National Recovery Plan for the Vancouver Island Marmot (Marmota vancouverensis) 2000 Update: RENEW Report No.19. Ottawa, Ontario: Environment Canada. Jule, K. R., Leavor, L. A., & Lea, S. E. (2008). The effects of captive experience on reintroduction survival in carnivores: A review and analysis. Biological Conservation 141, 355-363. Kreger, M. D., Hatfield, J. S., Estevez , I., Gee, G. F., & Clugston , D. A. (2005). The effects of captive rearing on the behavior of newly-released whooping cranes (Grus americana). Applied Animal Behavior Science 93, 165-178. Leighton, F. A. (2002). Health risk assessment of the translocation of wild animals. Rev. Sci. Tech. Off. Int. Epiz. 21(1) , 187-195. Mathews, F., Moro, D., Strachan, R., Gelling, M., & Buller, N. (2006). Health surveillance in wildlife reintroductions. Biological Conservation 131, 338-347. Matthews, F., Orros, M., McLaren, G., Gelling, M., & Foster, R. (2005). Keeping fit on the ark: assessing the suitability of captive-bred animals for release. Biological Conservation 121, 569-577. McPhee, M. E. (2003). Generations in captivity increases behavioral variance: considerations for captive breeding and reintroduction programs. Biological Conservation 115 , 71-77. Mellish, J. E., Hindle, A. G., & Horning, M. (2010). A preliminary assessment of the impact of disturbance and handling on Weddell seals of McMurdo Sound, Antarctica. Antarctic Science 22 (1), 25-29. 18 Nagorsen, D. W., Keddie, G., & Luszcz, T. (1996). Vancouver Island Marmot Bones from Subalpine Caves: Archaeological and Biological Significance, Occasional Paper No. 4. Victoria, British Columbia: Ministry of Environment, Lands and Parks. Ostermann, S. D., Deforge, J. R., & Edge, W. D. (2001). Captive breeding and reintroduction evaluation criteria: a case study of peninsular bighorn sheep. Conservation Biology 15(3), 749-760. Robert, A. (2009). Captive breeding genetics and reintroduction success. Biological Conservation 142, 2915-2922. Robert, K. A., & Schwanz, L. E. (2013). Monitoring the health status of free-ranging Tammar wallabies using hematology, serum biochemestry, and parasite loads. The Journal of Wildlife management 77 (6), 1232-1243. Rout, T. M., Hauser, C. E., & Possingham, H. P. (2007). Minimize long-term loss or maximize short-term gain? Optimal translocation strtaegies for threatened species. Ecological Modelling 201 , 67-74. Ryser-Degiorgis, M.-P. (2013). Wildlife health investigations: needs, challenges and recommendations. BMC Veterinary Research 9 9223). Sackett, L. C., Collinge, S. K., & Martin, A. P. (2013). Do pathogens reduce genetic diversity of their hosts? Variable effects of sylvatic plague in black-tailed prairie dogs. Molecular Ecology 22, 2441-2455. Snyder, N. F., Derrickson, S. R., Bessinger, S. R., Wiley, J. W., Smith, T. B., Toone, W. D., & Miller, B. (1996). Limitations of captive breeding in endangered species recovery. Conservation Biology 10 (2), 338-348. Stevenson, R. D., & Woods, Jr., W. A. (2006). Condition indices for conservation: new uses for evolving tools. Integrative and Comparative Biology 46 (6), 1169-1190. Swarth, H. S. (1911). Two new species of marmots from North America. University of California Publications in Zoology (7), 201-204. Swarth, H. S. (1912). Report on a collection of birds and mammals from Vancouver Island. University of California Publications in Zoology (10), 1-124. Teixeira, C. P., De Azevedo, C. S., Mendl, M., Cipreste, C. F., & Young, R. J. (2007). Revisiting translocation and reintroduction programmes: the importance of considering stress. Animal Behaviour 73, 1-13. 19 Vancouver Island Marmot Captive Management Group (2017). Vancouver Island Marmot Captive Management Group Meeting Minutes and Annual Summary. Internal Report, Vancouver Island Marmot Recovery Team. Unpublished. Vancouver Island Marmot Recovery Team (2017). Recovery Plan for the Vancouver Island Marmot in British Columbia. Victoria, B.C.: B.C. Ministry of Environment. Vancouver Island Marmot Recovery Team (2008). Recovery Strategy for the Vancouver Island Marmot (Marmota vancouverensis). Victoria, B.C.: B.C. Ministry of Environment. Villers, L. M., Jang, S. S., Lent, C. L., Sock-Cheng, L.-K., & Norosoarinaivo, J. A. (2008). Survey and comparison of major intestinal flora in captive and wild ring-tailed lemur (Lemur catta) populations. American Journal of Primatology 70, 175-184. Vitali, S., Reiss, A., & Eden, P. (2011). Conservation medicine in and through zoos. International Zoo Yearbook 45, 160-167. Williams, S. E., & Hoffman, E. A. (2009). Minimizing genetic adaptation in captive breeding programs: A review. Biological Conservation 142, 2388-2400. Woodford, M. H. (1993). International disease implications for wildlife translocations. Journal of Zoo and Wildlife Medicine 24(3), 265-270. 20 CHAPTER 2 HEMATOLOGICAL AND SERUM BIOCHEMICAL PARAMETERS IN THE VANCOUVER ISLAND MARMOT (MARMOTA VANCOUVERENSIS): REFERENCE RANGES AND A COMPARISON OF VALUES BETWEEN MANAGEMENT GROUPS INTRODUCTION The Vancouver Island marmot (Marmota vancouverensis) is a critically endangered member of the family Sciuridae, endemic to the subalpine meadows of central Vancouver Island, located off the southwest coast of British Columbia, Canada (Swarth, 1912; Swarth, 1911). In the late 1980s this rare species started to exhibit serious population declines (Bryant & Page, 2005). By 1998 marmot numbers had dropped below 100, and by 2003 there were fewer than 30 wild individuals (Jackson et al. 2015; Janz et al. 2000). In response to this dramatic attrition, a concerted captive breeding and reintroduction program was initiated in 1997. Since its inception, this program has included the participation of the Toronto Zoo (TZ, 1997 to present), the Calgary Zoo (CZ, 1998 to present), and the Mountain View Conservation and Breeding Society in Langley, British Columbia (MV, 2000 to 2014). In 2001 a purpose-built, quarantine and breeding facility, the Tony Barrett Mount Washington Marmot Recovery Centre (MRC), became operational at Mount Washington, Vancouver Island. As of December 2017, 660 marmots have been associated with the captive program, including 63 wild-born individuals originally captured for breeding purposes and 597 marmots born and weaned in captivity. Reintroductions of captive animals began in 2003 and by 2017 a total of 501 marmots (8 of the original wildborn and 493 of the captive-born) or 75.9% of the overall captive total, have been 21 released to the wild. Although these recovery efforts have helped to increase the wild population from its low point in 2003, conservation of the Vancouver Island Marmot (VIM) continues to involve an intensive program of ex situ management, reintroductions, and translocations (Vancouver Island Marmot Recovery Team, 2017). The health of an endangered species like the VIM can be negatively affected by many biotic and abiotic factors, including environmental perturbations, low population numbers, and artificial manipulation (Jackson et al. 2015; Moorhouse et al., 2006). Although monitoring health and changes in health is important for the vigilent recognition, ellucidation, and mitigation of potential threats that may jeopardise species viability and recovery, effective health surveillence represents a significant challenge for conservation programs (Jackson et al. 2015; Robert & Schwanz, 2013; Delahay et al., 2009; Miller, 2007; Daszak, et al. 2001; Snyder, et al. 1996). Many studies characterising wildlife health have emphasized dysfunction, pathogens, and mortality in populations (Ryser-Degiorgis, 2013). However, the ability to recognize and contextualize abnormality or disease requires an appreciation of ‘normal’ (Dimauro, et al., 2008). Baseline measurements of health parameters, often lacking in many wildlife species (Maceda-Veiga, et al., 2015), can be used to delineate variability and to compare what is expected for a species under different management regimes. This in turn may identify determinants and possible risk factors that are influencing health in individuals and populations (Hanisch, et al. 2012; Vitali et al., 2011; Mathews et al., 2006; Deem et al. 2001). A wide variety of morphological, physiological, biochemical, and condition indices have been used to quantify health and fitness at both the individual and population level (Peig & Green, 2009; Stevenson & Woods, Jr., 2006; Wisely, et al., 2005). This includes 22 hematology and serum biochemistry, which have been used to describe and compare physiological state, adaptation to different habitat conditions, nutritional status, body condition, organ function, relative stress, immune status, biological integrity, the presence of infectious or non-infectious disease, inflammation, and parasite burdens (Ruykys et al., 2012; Mellish et al., 2010; Moorhouse et al., 2007; Masello & Quillfeldt, 2004). In this project, VIM hematology and serum biochemistry metrics were analysed to delineate expected values and their range of variability in clinically normal animals. Hematology and serology parameters also were compared between different management conditions (wild, captive and captive-release, un-implanted and implanted). The goals of this project were to compile the first set of hematology and serum reference values for the VIM and assess the potential role of these parameters for comparing and monitoring health in this species. MATERIALS AND METHODS Classification of marmot management groups 1. Captive marmots were defined as any captive-born marmot originating from one of the four captive facilities or any wild-born marmot that had been captured for the captive breeding program and had therefore spent at least one hibernation in any of these captive facilities. 2. Captive-release marmots were animals released to the wild from captivity, including those originally born in captivity and any wild-born marmot that had been maintained in captivity for more than a single active season prior to release. 3. Wild marmots were wild-born individuals that had never been maintained in a captive facility or any wild-born marmot that had been temporarily maintained at the MRC for less than a single, active season and then returned to the wild. 23 Captive-release and wild marmots were collectively defined as free-ranging and with a few exceptions were surgically implanted with a very high frequency (VHF) radio-transmitter (Appendix E). Classification of marmot ages Marmot ages were categorized as young-of-the-year (pups or juveniles in their first summer), yearling (individuals in their second summer), two-year old (third summer), and adult (fourth or subsequent summers). The age of all captiveborn individuals was known with certainty. The age categories of wild-born marmots were known or extrapolated from previously described pelage characteristics (Bryant 1998) and relative size and appearance. In the field, age categories could be determined with relative certainty, except in the case of some individuals identified as two year-olds that exhibited characteristics which overlapped with those of yearlings and adults. Wild adults that were handled and identified for the first time could only be categorized as being three years of age or older, due to the non-specificity of their size and pelage characteristics. Sample collection and analysis Comprehensive physical examinations were conducted on all marmots at the time of blood collection. These examinations were conducted by a veterinarian and routinely involved an evaluation of the cardiovascular, respiratory, musculoskeletal, nervous, integumentary, and urogenital systems and an assessment of the marmot’s body condition. Marmots that did not display identifiable abnormalities at the time of examination were classified as clinically normal. For the purposes of this analysis, blood samples collected from captive marmots with pre-existing or newly identified health concerns at the time of sampling and those from quarantined, wild-caught marmots undergoing the chronic stress associated with transitioning to captive conditions (Cabezas et al., 2007), were categorized as clinically abnormal. These 24 clinically abnormal samples were excluded from calculation of the reference values and in analyses comparing age, sex, and management groups. They only were used to explore the possibility that there were group differences between clinically normal and abnormal animals. Samples were collected in captivity or at field sites in association with the following circumstances (Figure 2.1): A. Captive Marmots: i. Annual or biennial health evaluation of captive marmots maintained at the TZ, CZ, MV and MRC (258 individuals, 606 samples). These marmots did not have implanted abdominal radio-transmitters at the time of sampling (un-implanted). ii. Pre-operative evaluation of captive marmots prior to surgical implantation with abdominal radio-transmitters (188 individuals, 188 samples). These marmots did not have implants at the time of sampling. Categories i. and ii. (healthy, un-implanted, captive marmots) represented the largest number of samples, accounting for 76.4% of the overall total. iii. Evaluation of implanted, captive-release marmots on the day of release or on the day that preceded it (133 individuals, 133 samples). Following surgery, captive, implanted marmots were afforded a period of convalescence in captivity before being released to the wild. The interval between captive surgery and release ranged from 11 to 93 days (average = 28 days). iv. Examinations that identified or addressed specific health concerns in captive marmots (30 individuals, 44 samples). Categories of health concerns included congenital or early onset problems, infections / inflammation, heart disorders, neoplasia, and trauma. 25 v. Health evaluation of marmots during quarantine. These wild-caught marmots were being transitioned into the captive program (23 individuals, 23 samples). Samples from (iv.) and (v.) were categorized as clinically abnormal. B. Free-ranging Marmots: vi. Evaluation of implanted, post-release marmots prior to surgical replacement of radio-transmitters (16 individuals, 16 samples). vii. Pre-operative evaluation of wild marmots prior to surgical implantation with abdominal radio-transmitters (66 individuals, 66 samples). Marmots did not have an implanted radio-transmitter at the time of sampling. viii. Evaluation of implanted, wild marmots prior to replacement of radiotransmitters (14 individuals, 19 samples). ix. Health evaluation of implanted, wild marmots which were opportunistically recaptured for evaluation or translocation (11 individuals, 11 samples). 26 Figure 2.1. Schematic diagram illustrating the relationships and sample size of the Vancouver Island Marmot (Marmota vancouverensis) blood sampling categories used in the analyses. “Implanted” refers to individuals with previously implanted radio-transmitters. The arrows indicate groups with the potential for overlap (i.e. an individual may have been sampled as part of a pre-surgical examination and then again prior to release, or at the time of recapture or radio-transmitter replacement). CLINICALLY NORMAL (N = 1,039 samples) CLINICALLY ABNORMAL (N = 67 samples) A. CAPTIVE (994 samples) (iii) IMPLANTED (133) (I & ii) UN-IMPLANTED (794) (iv) HEALTH CONCERN (44) (v) QUARANTINE (23) B. FREE RANGING (112) CAPTIVE RELEASE (16) (vi) IMPLANTED (16) WILD (96) (viii & ix) IMPLANTED (30) (vii) UN-IMPLANTED (66) IMPLANTED (179) UN-IMPLANTED (860) CAPTURED FOR CAPTIVITY 26 27 Whole blood samples (up to 6 millilitres in volume) were obtained following immobilization with an intramuscular injection of ketamine hydrochloride combined with midazolam hydrochloride (various manufacturers) at approximately 10 mg/kg and 0.25 mg/kg, respectively and then maintained on inhaled isoflurane. In some instances, marmots were mask induced with isoflurane without receiving any injectable immobilization agents (Graham Crawshaw, Toronto Zoo, personal communication). Blood samples were collected from the cephalic, saphenous, femoral, or tarsal veins. Following venipuncture, a portion of the whole blood was promptly transferred into a vacuum tube containing the anticoagulant ethylenediamine-tetraacetic acid (EDTA) and into a serum-separating vacuum tube (SST) containing gel. The SST samples were allowed to clot, and then centrifuged. A small portion of whole blood was used to make two blood smears, which were subsequently airdried at room temperature or in the field, at ambient temperature. Captive and field samples were kept cool and protected from heat, direct sunlight, and freezing temperatures. The data used in this analysis were derived from samples collected over a 23-year period from marmots at geographically disparate locations. Because of lab changeover and logistical practicalities, samples were submitted to one of four diagnostic laboratories; the in-house laboratory at the Toronto Zoo (n=122), Central Laboratory for Veterinarians in Calgary, Alberta or Langley, British Columbia (combined, n=648) and Idexx Reference Laboratories in Delta, British Columbia (n=268). Ninety-eight per cent or 1,084 of the submitted samples were analyzed within three days of collection. The 16 hematological parameters included total white blood cells (WBC), red blood cells (RBC), hemoglobin (Hb), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin 28 concentration (MCHC), red cell distribution width (RDW), platelet count, mean platelet volume, segmented neutrophils, band neutrophils, lymphocytes, monocytes, eosinophils and basophils. The 30 serological parameters included glucose, blood urea nitrogen (BUN), creatinine, BUN / creatinine ratio, sodium (Na), potassium (K), sodium / potassium ratio, chloride (Cl), bicarbonate, carbon dioxide, anion gap, calcium, phosphorous (P), calcium / phosphorous ratio, total protein, albumin, globulin, albumin / globulin ratio (A/G ratio), total bilirubin, alkaline phosphatase (ALP), alanine amino transferase (ALT), aspartate amino transferase (AST), gamma glutamyl transferase (GGT), creatinine phosphokinase (CK), calculated osmolality, lactose dehydrogenase (LDH), amylase, lipase, cholesterol and tetra iodothyronine. In keeping with management activities, all blood samples were collected during the marmots’ active season. Although the blood parameters of other true hibernators have been found to exhibit changes during hibernation, including decreases in leukocytes, total bilirubin, glucose, and creatinine, and increases in total protein and albumin (Feoktistova et al., 2016), these trends could not be investigated with the availible data. Free-ranging marmots were not accessible for sampling during the hibernation period. Although there was the potential to sample captive marmots, which were allowed to hibernate as a standard management protocol, this did not occur due to concerns about prolonged clotting times associated with physiological changes during torpor, such as thrombocytopenia (Cooper et al., 2012). Baseline reference values were calculated in accordance with guidelines established by Species 360 (formerly the International Species Information System), which incorporate several steps to minimize the potential influence of multiple laboratories and repeated sampling of the same individuals (Teare, 2002). In accordance with these guidelines, the mean and standard deviation are calculated for each of the hematological and serological variables generated from clinically 29 normal or healthy animals. The standard deviation for these variables was multiplied by three, and the resulting value (representing 3 standard deviations or 99.7% of the values) was subtracted and added to the calculated mean. Any values lying outside this calculated lower and upper range were deemed to be outliers and were removed. The mean and standard deviation were then re-calculated without the outliers (Teare, 2002; de With et al., 1999). For the VIM baseline reference values, all clinically normal marmots from all groups, including captive, captive-release, and wild (inclusive of implanted and non-implanted animals) were included in the analysis. Subsequent analyses focused on comparing the major management groups, specifically wild, captive, and captive-release marmots. Values for clinically normal marmots without abdominal radio-transmitters were compared to clinically normal, implanted marmots and to values for marmots in the clinically-abnormal group. Data were visually examined using dot plots and box plots to identify potential trends or differences with respect to marmot age, sex, and season. Statistical analyses were performed using R (version 3.3.1, R Development Project, https://www.r-project.org). Normality of data distribution was assessed visually using histograms. Statistical comparison of parameters was performed using Student’s unpaired t-test. All tests were two-tailed and statistical significance was assigned at α = 0.01. RESULTS From August 1992 to June 2015, 1,106 blood samples were opportunistically collected from 439 (243 males, 196 females) captive, captive-release, and wild VIM. Individual marmots were sampled between 1 and 14 times. The overall average was 2.52 samples per individual marmot. The sampling mean for captive marmots was 30 2.85 (range 1 to 14) and for wild individuals it was 1.06 (range 1 to 4). Sixty-seven of the samples were categorized as clinically abnormal. The remaining 1,039 blood samples were obtained from a total of 429 individual marmots determined to be clinically normal or healthy at the time of sampling. Blood sample distribution with respect to year of collection is presented in Figure 2.2. The blood samples were collected over a 23 year-period, indicating the potential for differences with respect to technicians, laboratories and analytical techniques over time. Monthly distribution of sample collection is presented in Figure 2.3. All blood samples were collected during the marmots’ active season with over 90% being obtained between June and September. Most of the routinely scheduled annual or biennial health evaluations of captive marmots, which represented the largest category of blood samples, occurred in September, and this is reflected in the graph. The sex-age distribution of sampled marmots is presented in Figure 2.4. Although there is relatively comparable representation of males and females, the sample set was biased towards younger individuals. The age distributions of freeranging and captive samples are compared in Figure 2.5. The greater longevity and accessibility of captive marmots allowed more opportunities for collection of blood samples from older animals, in contrast to their free-ranging counterparts Hematology and serum biochemistry reference values derived from all clinically normal VIM and following Species 360 guidelines are presented in Tables 2.1 and 2.2. The means of VIM reference values are compared to values that have been generated for other small to medium sized rodents in Table 2.3. The general characteristics of the VIM reference ranges appear to be qualitatively comparable to values that have been generated for these other species of rodents. 31 Figure 2.2. Annual distribution of 1,106 blood samples collected from Vancouver Island Marmots (Marmota vancouverensis) 1992 to 2015. Number of samples 250 200 150 100 50 0 1992 1994 1996 1998 2000 2002 2004 2006 2008 2010 2012 2014 Year Figure 2.3. Monthly distribution of 1,106 blood samples collected from captive and free-ranging Vancouver Island Marmots (Marmota vancouverensis). Number of samples 500 400 Captive (n = 998) 300 Free-ranging (n = 108) 200 100 0 Jan Feb Mar Apr May Jun Jul Month Aug Sept Oct Nov Dec 32 Figure 2.4. Sex and age distribution of 1,106 blood samples collected from captive Number of samples and free-ranging Vancouver Island Marmots (Marmota vancouverensis). 180 160 140 120 100 80 60 40 20 0 Male (n = 596) Female (n = 510) 0 1 2 3 4 5 6 7 8 Age (years) 9 10 11 12 13 Figure 2.5. Comparison of age distribution of 1,106 blood samples collected from captive and free-ranging Vancouver Island Marmots (Marmota vancouverensis). Number of samples 300 250 Captive (n = 998) 200 Free-ranging (n = 108) 150 100 50 0 0 1 2 3 4 5 6 7 8 Age (years) 9 10 11 12 13 33 Table 2.1. Reference intervals for 16 hematological parameters from clinically healthy Vancouver Island Marmots (Marmota vancouverenis). Parameter Units Mean Min Max N S.D. White blood cells x 10⁹/L 4.96 0.60 12.20 1018 1.89 Segmented neutrophils x 10⁹/L 2.64 0.25 7.56 1017 1.20 Band neutrophils x 10⁹/L 0.00 0.00 0.00 1012 0.00 Lymphocytes x 10⁹/L 1.93 0.01 5.55 1018 1.13 Monocytes x 10⁹/L 0.31 0.00 1.40 1012 0.25 Eosinophils x 10⁹/L 0.03 0.00 0.25 1011 0.05 Basophils x 10⁹/L 0.01 0.00 0.12 1003 0.02 Red blood cells x 10 ¹²/L 6.44 4.2 8.49 1010 0.63 Hemoglobin g/L 145.10 98 187 1004 14.32 Hematocrit L/L 0.43 0.29 0.557 1021 0.04 fl 65.90 50 82.3 977 3.51 pg 22.44 16.3 33.1 986 1.60 g/L 339.29 294 384 983 12.26 %CV 14.35 10.1 20.3 877 1.52 x 10⁹/L 318.70 37 625 761 91.68 fl 8.91 6.7 14 694 1.26 WHITE BLOOD CELLS ERYTHROCYTES Mean corpuscular volume Mean corpuscular hemoglobin Mean corpuscular hemoglobin concentration Red cell distribution width HEMOSTASIS Platelet count Mean platelet volume 34 Table 2.2. Reference intervals for 30 serum biochemistry parameters from clinically healthy Vancouver Island Marmots (Marmota vancouverensis). Parameter Units Mean Min Max N S.D. Sodium mmol/L 143.84 130 155.8 525 4.05 Potassium mmol/L 5.37 3.3 13.3 522 1.42 Sodium / Potassium ratio ratio 28.14 8.6 43.6 525 6.08 Chloride mmol/L 102.11 88 112 528 4.11 Bicarbonate mmol/L 26.37 9 39 147 5.88 Carbon Dioxide mmol/L 26.37 5 38 357 5.60 20.71 6 41 512 6.56 ELECTROLYTES AND ACID-BASE Anion Gap Calcium mmol/L 2.39 1.44 3.33 598 0.21 Phosphorous mmol/L 1.83 0.66 3.48 594 0.47 Calcium / Phosphorous ratio ratio 1.39 0.44 4.25 573 0.41 Calculated Osmolality mmol/kg 297.25 276 319 508 6.73 Total Protein g/L 61.97 42 90 643 8.97 Albumin g/L 27.30 17 43 555 3.56 Globulin g/L 32.85 18.6 53 553 6.03 Albumin / Globulin ratio ratio 0.86 0.3 1.9 561 0.20 Total Bilirubin μmol/L 2.63 0 7 428 1.37 Alkaline Phosphatase IU/L 71.58 5 266 556 41.35 IU/L 18.64 1 117 512 16.97 IU/L 37.08 1 222 449 34.51 IU/L 3.96 0 18 516 3.01 PROTEINS LIVER AND MUSCLE Alanine Aminotransferase Aspartate Aminotransferase Gamma Glutamyltransferase 35 Table 2.2. (cont.) Reference intervals for 30 serum biochemistry parameters from clinically healthy Vancouver Island Marmots (Marmota vancouverensis). Parameter Units Mean Min Max N S.D. Creatinine Phosphokinase IU/L 526.24 56 2970 583 366.23 Cholesterol mmol/L 7.24 3.71 13.05 72 1.98 Glucose mmol/L 8.78 2.2 15.8 630 2.29 Blood Urea Nitrogen mmol/L 10.92 3.1 22.2 632 2.60 Creatinine μmol/L 81.91 18 150 617 21.80 Bun / Creatinine ratio ratio 0.14 0.025 0.376 613 0.06 Lactose Dehydrogenase IU/L 1784.97 865 4068 30 822.29 Amylase IU/L 839.79 147 2086 77 423.02 Lipase IU/L 191.79 60 856 73 137.25 Tetra Iodothyronine nmol/L 57.17 20 91 151 16.82 LIVER AND MUSCLE RENAL FUNCTION OTHER 36 Table 2.3. Vancouver Island Marmot (Marmota vancouverenis) hematological and serological mean values compared to published means for the Woodchuck (Bellezza, et al., 2015) and normal values published for Mice (Mus musculus), Rats (Rattus norvegicus), Gerbils (Meriones unguiculatus), and Guinea Pigs (Cavia porcellus) (Harkness & Wagner, 1995). Units VIM mean Woodchuck low / high mean (a) Mouse range Rat range Gerbil range Guinea Pig range x 10⁹/L 5 8.7 / 10.4 6 - 15 6 - 17 7 - 15 7 - 18 Segmented neutrophils % 53 63 (b) 10 - 40 9 - 34 5 - 34 28 - 44 Lymphocytes % 39 26 (b) 55 - 95 65 - 85 60 - 95 39 - 72 Monocytes % 6 6 (b) 0.1 - 3.5 0-5 0 -3 3 - 12 Eosinophils % 0.6 4 (b) 0-4 0-6 0-4 1-5 Basophils % 0.2 0 - 0.3 0 - 1.5 0-1 0-3 Red blood cells x 10 ¹²/L 6.4 4.7 / 5.3 7.0 - 12.5 7 - 10 8-9 4.5 - 7 Hemoglobin g/L 145 122 / 132 102 - 166 110 - 180 126 - 162 110 - 150 Hematocrit L/L 0.43 0.36 / 0.41 0.39-0.49 0.36-0.48 0.43-0.49 0.37-0.48 Mean corpuscular volume fl 66 73 / 77 Mean corpuscular hemoglobin pg 22 25 / 26 Mean corpuscular hemoglobin concentration g/L 340 340 / 360 Red cell distribution width %CV 14 14 / 17.5 Parameter WHITE BLOOD CELLS White blood cells ERYTHROCYTES (a) High and low means arising from multiple studies as reported in Bellezza, et al., 2015 (b) Published values in which the collective mean of the differential count equals the mean of the total white blood cells 37 Table 2.3. (cont.) Vancouver Island Marmot (Marmota vancouverenis) hematological and serological mean values compared to published means for the Woodchuck (Bellezza, et al., 2015) and normal values published for Mice (Mus musculus), Rats (Rattus norvegicus), Gerbils (Meriones unguiculatus), and Guinea Pigs (Cavia porcellus) (Harkness & Wagner, 1995). Units VIM mean Woodchuck low / high mean (a) Mouse range Rat range Gerbil range Guinea Pig range x 10⁹/L 319 451 / 525 800-1100 500-1300 400 - 600 250 - 850 fl 8.9 6.8 / 7.1 Sodium mmol/L 144 143 / 151 112 - 193 135 - 155 144 - 158 132 - 156 Potassium mmol/L 5.4 3.7 / 4.7 5.1 - 10.4 4-8 3.8 - 5.2 4.5 - 8.9 Sodium / Potassium ratio ratio 28 34 / 40 Chloride mmol/L 102 97 / 102 82 - 114 94 - 116 93 - 118 98 - 115 Bicarbonate mmol/L 26 30 / 34 Carbon Dioxide mmol/L 26 31 / 34 20 15 / 20 Parameter HEMOSTASIS Platelet count Mean platelet volume ELECTROLYTES AND ACIDBASE Anion Gap Calcium mmol/L 2.4 2.3 / 2.6 0.8 - 2.1 1.3 - 3.2 0.9 - 1.6 1.3 - 3.0 Phosphorous mmol/L 1.8 1.2 / 1.7 0.7 - 3.0 1.7 - 2.7 1.2 - 2.3 1.0 - 3.9 Total Protein g/L 62 55 / 69 35 - 72 56 - 76 43 - 125 46 - 62 Albumin g/L 27 23 / 37 25 - 48 38 - 48 18 - 55 21 - 39 Globulin g/L 33 30 / 36 6 18 - 30 12 - 60 17 - 26 Albumin / Globulin ratio ratio 0.9 0.7 / 1.1 PROTEINS (a) High and low means arising from multiple studies as reported in Bellezza, et al., 2015 38 Table 2.3. (cont.) Vancouver Island Marmot (Marmota vancouverenis) hematological and serological mean values compared to published means for the Woodchuck (Bellezza, et al., 2015) and normal values published for Mice (Mus musculus), Rats (Rattus norvegicus), Gerbils (Meriones unguiculatus), and Guinea Pigs (Cavia porcellus) (Harkness & Wagner, 1995). Units VIM mean Woodchuck low / high mean (a) Mouse range Rat range Gerbil range Guinea Pig range Total Bilirubin μmol/L 2.6 1.9 / 5.5 1.71-15.4 3.4 - 9.4 3.4 - 10.3 5.1 - 15.4 Alkaline Phosphatase IU/L 72 7.2 / 19.3 45 - 222 16 - 125 12 - 37 18 - 28 Alanine Aminotransferase IU/L 19 1.0 / 3.5 26 - 77 16 - 89 10 - 25 Aspartate Aminotransferase IU/L 37 21 / 34 54 - 269 192 - 262 45.5-48.2 Gamma Glutamyltransferase IU/L 4 1.7 (b) Creatinine Phosphokinase IU/L 526 478 / 690 Cholesterol mmol/L 7.2 3.6 / 5.4 0.7 - 2.1 1 - 3.4 2.3 - 3.9 0.5 - 1.1 Glucose mmol/L 8.8 10.2 / 12.1 3.4 - 9.7 2.8 - 7.5 2.8 - 7.5 3.3 - 7 Blood Urea Nitrogen mmol/L 11 4.6 / 9 4.3 - 10 5.4 - 7.5 6 - 9.6 3.2 - 11.3 Creatinine μmol/L 82 79.5 / 133 26 - 88 18 - 71 53 - 124 53 - 194 Amylase IU/L 840 2210 / 2645 Lipase IU/L 192 201 / 361 Parameter LIVER AND MUSCLE RENAL FUNCTION OTHER (a) High and low means arising from multiple studies as reported in Bellezza, et al., 2015 (b) One mean reported in Bellezza, et al., 2015 39 Seasonal Trends. Total white blood cells, neutrophils, lymphocytes, monocytes, total protein and albumin increased during the course of the active season (Figure 2.6), while platelet count, total bilirubin, glucose and creatinine did not exhibit change. In laboratory Woodchucks (Marmota monax), the hematocrit has been reported to be higher in the spring than in the autumn (Bellezza, et al., 2015), but this was not apparent from these data. Woodchucks and Yellow-bellied Marmots (Marmota flaviventris), also are reported to have an increase in red blood cells, hematocrit, and hemoglobin from early to late summer in preparation for hibernation (Armitage, 2014). VIM exhibited a similar trend in all three parameters from June to October, but the magnitude of the difference was relatively small (Figure 2.7). Age trends. Red blood cells, hematocrit, and hemoglobin appeared to decline with marmot age (Figure 2.8) whereas the leukogram (including total white blood cells, neutrophils, lymphocytes, monocytes, eosinophils, and basophils) showed no age pattern. Glucose decreased with age whereas amylase increased with age (Figure 2.9). Elevation trends. Acclimatization to lower elevations has resulted in decreased hematocrit levels in Yellow-bellied Marmots (Armitage, 2014). The mean of hematocrit values from captive marmots maintained at the MV (elevation 25 meters) were 5.5, 5.6, and 7.6% lower than respective values from the CZ (elevation 1027 meters), TZ (elevation 145 meters), and the MRC (elevation 1244 meters), and these differences were statistically significant (P < 0.0001). However, there was no significant difference or discernable elevation trend between the other three institutions and this could indicate that the MV values are the result of some facility effect rather than elevation. Sex trends. In several species, female hematocrit values are lower than those of males (Probst, et al., 2006). A sex difference in hematocrit was seen in these data and 40 was statistically significant (male mean = 0.434, female mean = 0.416, P < 0.0001). Amylase was lower in males than females (male mean = 711.11, female mean = 933.10, P = 0.0408) (Figure 2.10). There was no apparent or statistically-significant sex difference with respect to white blood cells (P = 0.4014) or glucose (P = 0.1779). Group comparisons. Parameters for clinically normal captive, wild, and captiverelease marmots are compared in Tables 2.4 and 2.5. Values for non-implanted, implanted, and clinically abnormal marmots are compared in Table 2.6 and 2.7. The statistical and clinical significance of differences between hematology and serum biochemistry parameters with respect to specific groups are summarised in Table 2.8 and 2.9. Eleven of the 46 parameters showed no clinical or statistical differences between any of the groups that were compared, including values which were derived from clinically abnormal animals. This included 2 hematology parameters; band neutrophils and eosinophils, and 9 serum parameters; anion gap, calcium, calculated osmolality, aspartate aminotransferase, gamma glutamyltransferase, creatinine phosphokinase, BUN / creatinine ratio, lactose dehydrogenase (data limited for some groups), and lipase (data limited for some groups). Twenty-eight of the 46 parameters showed evidence of statistically significant differences between certain groups, but the small magnitude and nature of these differences did not suggest physiological or clinical significance. Eight of the 46 parameters had biologically plausible and statistically significant differences between some of the compared groups (Table 2.8). This included 5 leukogram parameters; white blood cells, segmented neutrophils, lymphocytes, monocytes, basophils, and 3 serum biochemistry parameters; total protein, albumin, and globulin. Potentially significant differences in leukogram and protein parameters occurred between the captive and free-ranging groups (both 41 wild and captive-release). Also, leukogram parameters of clinically normal, unimplanted marmots differed from the clinically abnormal group. 42 Figure 2.6. Changes in total white blood cells and total protein from April to October for clinically normal Vancouver Island Marmots (Marmota vancouverensis). Total Protein versus Julian Day 100 80 60 40 20 Total Protein (g/L) Total Protein (g/L) 30 25 20 15 10 5 0 WBC White Blood Cells (x 10⁹/L) 35 White Blood Cells versus Julian Day 150 200 Julian Day Julian Day 250 300 150 200 250 300 Julian Day Julian Day 42 43 Hemoglobin (g/L) Red Blood Cells (x 10 ¹²/L) Figure 2.7. Boxplot comparisons of red blood cells and hemoglobin between June and October for clinically normal Vancouver Island Marmots (Marmota vancouverensis). June October Month June October Month 43 44 Figure 2.8. Change in red blood cells and hemoglobin for clinically normal Vancouver Island Marmots (Marmota vancouverensis) according to age. Red Blood Cells versus Age Hemoglobin (g/L) 150 50 100 Hemoglobin (g/L) 7 6 5 4 3 Red Blood Cells Red Blood Cells (x 10⁹/L) 8 9 200 Hemoglobin versus Age 0 2 4 6 Age (years) Age (years) 8 10 12 0 2 4 6 8 10 12 Age (years) Age (years) 44 45 Figure 2.9. Change in glucose and amylase for clinically normal Vancouver Island Marmots (Marmota vancouverensis) according to age. Amylase versus Age 1500 1000 Amylase (IU/L) Amylase (IU/L) 10 500 5 Glucose (mmol/L) Glucose (mmol/L) 15 2000 Glucose versus Age 0 2 4 6 (years) AgeAge (years) 8 10 12 0 2 4 6 8 10 12 Age (years) Age (years) 45 46 Hematocrit Amylase (IU/L) Figure 2.10. Boxplots comparing male and female values for hematocrit and amylase in Vancouver Island Marmots (Marmota vancouverensis). Male Female Gender Female Male Gender 46 47 Table 2.4. Hematology values comparing healthy captive (un-implanted), wild and captive-release Vancouver Island Marmots (Marmota vancouverensis). Bold indicates comparisons with statistical and potential clinical significance (a). Units Captive Mean S.D. N Wild mean S.D. N Captiverelease mean S.D. N White blood cells x 10⁹/L 5.506 2.619 601 4.401 2.376 94 3.825 1.000 16 Segmented neutrophils x 10⁹/L 2.949 1.803 598 2.973 1.805 94 2.841 0.808 16 Band neutrophils x 10⁹/L 0.004 0.032 598 0.000 0.000 94 0.000 0.000 16 Lymphocytes x 10⁹/L 2.102 1.268 598 1.161 0.992 94 0.618 0.204 16 Monocytes x 10⁹/L 0.404 0.415 598 0.194 0.164 94 0.304 0.454 16 Eosinophils x 10⁹/L 0.034 0.074 598 0.049 0.090 94 0.031 0.045 16 Basophils x 10⁹/L 0.016 0.038 598 0.010 0.021 94 0.028 0.037 16 Red blood cells x 10 ¹²/L 6.38 0.789 597 6.32 0.613 94 6.28 0.530 16 Hemoglobin g/L 145.206 17.999 587 146.36 14.308 94 145.00 12.329 16 Hematocrit L/L 0.43 0.048 601 0.43 0.043 94 0.43 0.039 16 Mean corpuscular volume fl 66.356 4.782 564 67.93 2.752 94 68.63 2.752 16 Mean corpuscular hemoglobin pg 22.614 1.846 557 23.22 1.192 94 23.08 0.728 16 Mean corpuscular hemoglobin concentration g/L 340.397 18.244 567 341.00 10.327 94 336.81 11.397 16 Red cell distribution width %CV 14.471 1.623 475 12.99 3.576 94 14.76 2.059 16 x 10⁹/L 322.683 121.355 341 312.93 108.046 92 302.06 51.177 16 fl 8.980 1.653 332 8.13 0.933 86 8.99 1.829 16 Parameter WHITE BLOOD CELLS ERYTHROCYTES HEMOSTASIS Platelet count Mean platelet volume (a) Potential clinical significance was considered if the magnitude of the difference in the parameter comparison had practical relevance with respect to physiological or clinical state. 48 Table 2.5. Serum biochemistry values comparing healthy, captive (un-implanted), wild and captive-release Vancouver Island marmots (Marmota vancouverensis). Bold indicates comparisons with statistical and potential clinical significance (a). Units Captive Mean S.D. N Wild mean S.D. N Captiverelease mean S.D. N Sodium mmol/L 144.16 4.811 335 142.05 4.148 79 139.45 6.729 11 Potassium mmol/L 5.241 2.771 334 7.26 3.043 79 9.98 5.900 11 Sodium / Potassium ratio ratio 29.43 5.842 334 22.13 6.779 79 16.91 6.039 11 Chloride mmol/L 101.32 6.094 332 103.10 4.205 79 99.27 4.650 11 Bicarbonate mmol/L 26.29 6.624 96 25.93 4.131 15 27.44 2.297 9 Carbon Dioxide mmol/L 26.66 5.979 211 24.33 5.673 64 28.50 3.536 2 21.39 7.333 318 21.65 7.968 79 22.55 4.712 11 Parameter ELECTROLYTES AND ACIDBASE Anion Gap Calcium mmol/L 2.40 0.222 404 2.42 0.979 79 2.28 0.109 11 Phosphorous mmol/L 1.70 0.427 405 2.14 0.740 79 1.82 0.526 11 Calcium / Phosphorous ratio ratio 1.50 0.385 379 1.26 0.709 79 1.34 0.364 11 mmol/kg 298.73 7.913 317 297.20 6.464 79 294.36 5.801 11 Total Protein g/L 65.55 9.919 455 54.00 6.278 79 55.18 6.080 11 Albumin g/L 29.09 4.833 369 25.68 7.185 79 23.45 3.078 11 Globulin g/L 35.29 7.131 365 29.01 4.648 78 31.73 3.636 11 Albumin / Globulin ratio ratio 0.90 0.619 366 0.88 0.139 79 0.75 0.082 11 Total Bilirubin μmol/L 2.81 1.848 263 3.16 1.718 64 4.56 1.236 9 Alkaline Phosphatase IU/L 75.70 60.877 367 94.48 123.009 79 75.00 26.680 11 Alanine Aminotransferase IU/L 28.35 40.501 333 17.46 12.506 79 13.09 7.739 11 Aspartate Aminotransferase IU/L 49.89 72.258 311 36.60 24.691 52 54.09 38.454 11 Gamma Glutamyltransferase IU/L 4.77 6.046 333 4.66 3.293 76 3.36 2.420 11 Creatinine Phosphokinase IU/L 649.92 987.135 398 813.01 1193.698 79 522.00 111.966 11 Calculated Osmolality PROTEINS LIVER AND MUSCLE (a) Potential clinical significance was considered if the magnitude of the difference in the parameter comparison had practical relevance with respect to physiological or clinical state. 49 Table 2.5. (cont.) Serum biochemistry values comparing healthy, captive (unimplanted), wild and captive-release Vancouver Island marmots (Marmota vancouverensis). Bold indicates comparisons with statistical and potential clinical significance (a). Parameter Units Captive Mean S.D. N Wild mean S.D. N Captiverelease mean S.D. N Cholesterol mmol/L 7.94 1.994 37 6.32 1.388 33 No data No data No data Glucose mmol/L 8.97 2.504 440 8.75 2.669 79 6.81 2.071 11 Blood Urea Nitrogen mmol/L 11.85 3.967 441 10.48 2.712 79 9.63 2.633 11 Creatinine μmol/L 83.31 24.741 427 73.59 22.758 79 65.55 10.211 11 Bun / Creatinine ratio ratio 0.16 0.086 427 0.16 0.067 79 0.151 0.052 11 Lactose Dehydrogenase IU/L 1784.97 822.289 30 No data No data No data No data No data No data Amylase IU/L 1105.81 496.744 43 587.21 279.358 34 No data No data No data Lipase IU/L 181.11 134.140 38 204.91 145.485 33 No data No data No data Tetra Iodothyronine nmol/L 61.88 14.315 77 42.50 14.470 46 No data No data No data RENAL FUNCTION OTHER (a) Potential clinical significance was considered if the magnitude of the difference in the parameter comparison had practical relevance with respect to physiological or clinical state. 50 S.D. N 5.213 1.970 174 854 2.909 1.428 0.027 854 0.000 1.978 1.237 854 x 10⁹/L 0.346 0.373 Eosinophils x 10⁹/L 0.035 Basophils x 10⁹/L Red blood cells S.D. N 6.468 3.718 65 174 3.726 2.583 65 0.000 174 0.000 0.000 65 1.936 1.169 174 2.111 1.406 65 854 0.311 0.283 174 0.505 0.673 65 0.075 854 0.035 0.062 174 0.037 0.069 65 0.015 0.035 854 0.019 0.036 174 0.041 0.152 65 x 10 ¹²/L 6.35 0.74 853 6.73 0.55 173 6.31 0.87 60 Hemoglobin g/L 143.47 16.94 843 148.87 11.07 174 138.35 20.54 60 Hematocrit L/L 0.42 0.05 857 0.44 0.03 174 0.41 0.06 64 Mean corpuscular volume fl 66.13 4.39 819 65.73 2.76 174 64.62 4.30 60 pg 22.49 1.68 813 22.17 1.11 174 21.95 1.45 60 g/L 340.06 16.18 823 337.01 9.87 174 340.05 14.49 60 %CV 14.24 2.14 731 14.62 2.01 174 14.68 3.17 57 x 10⁹/L 314.15 106.686 595 354.08 92.153 174 394.21 172.393 43 fl 8.92 1.500 570 8.94 1.204 174 8.61 1.500 40 Un-implanted mean Nonrepresentative mean Implanted mean Table 2.6. Hematology values comparing (i) clinically normal, un-implanted, (ii) clinically normal, implanted, and (iii) clinically abnormal Vancouver Island Marmots (Marmota vancouverensis). Bold indicates comparisons with statistical and potential clinical significance (a). S.D. N 5.081 2.447 857 x 10⁹/L 2.703 1.642 Band neutrophils x 10⁹/L 0.003 Lymphocytes x 10⁹/L Monocytes Parameter Units White blood cells x 10⁹/L Segmented neutrophils WHITE BLOOD CELLS ERYTHROCYTES Mean corpuscular hemoglobin Mean corpuscular hemoglobin concentration Red cell distribution width HEMOSTASIS Platelet count Mean platelet volume (a) Potential clinical significance was considered if the magnitude of the difference in the parameter comparison had practical relevance with respect to physiological or clinical state. 51 S.D. N 143.35 4.31 72 460 6.74 3.53 6.08 460 24.77 101.86 5.62 458 mmol/L 26.48 6.33 mmol/L 26.46 S.D. N 144.19 3.82 54 72 5.38 1.15 54 7.86 72 27.78 5.86 52 102.58 4.63 72 101.23 3.58 52 117 25.97 3.67 30 25.87 4.67 15 5.67 316 25.64 4.97 42 25.88 8.29 33 20.78 7.06 444 21.73 7.50 72 22.71 8.72 48 Un-implanted mean Clinically abnormal mean Implanted mean Table 2.7. Serum biochemistry values comparing clinically normal un-implanted and implanted and clinically abnormal Vancouver Island Marmots (Marmota vancouverensis). Bold indicates comparisons with statistical and potential clinical significance (a). S.D. N 143.79 4.69 461 mmol/L 5.46 2.67 Sodium / Potassium ratio ratio 28.38 Chloride mmol/L Bicarbonate Carbon Dioxide Parameter Units Sodium mmol/L Potassium ELECTROLYTES AND ACID-BASE Anion Gap Calcium mmol/L 2.44 0.92 530 2.48 0.72 72 2.48 0.18 56 Phosphorous mmol/L 1.82 0.51 531 2.15 0.72 72 2.00 0.46 56 Calcium / Phosphorous ratio ratio 1.49 1.38 505 1.26 0.60 72 1.29 0.42 54 Calculated Osmolality mmol/kg 297.58 7.91 443 297.17 6.10 72 298.24 7.48 49 Total Protein g/L 62.89 10.47 581 56.72 5.53 72 60.59 7.92 58 Albumin g/L 28.04 4.72 495 26.06 7.47 72 26.89 4.36 55 Globulin g/L 33.50 7.22 491 31.47 4.22 71 33.42 6.96 55 Albumin / Globulin ratio ratio 0.90 0.54 492 0.81 0.13 72 0.85 0.22 53 Total Bilirubin μmol/L 2.70 1.73 370 3.08 1.51 66 3.44 3.18 48 Alkaline Phosphatase IU/L 75.14 55.83 493 90.07 125.44 72 62.24 38.63 54 Alanine Aminotransferase IU/L 24.46 35.46 455 14.68 11.50 72 18.94 53.38 51 Aspartate Aminotransferase IU/L 45.51 64.45 405 31.83 23.88 54 91.32 361.58 28 PROTEINS LIVER AND MUSCLE (a) Potential clinical significance was considered if the magnitude of the difference in the parameter comparison had practical relevance with respect to physiological or clinical state. 52 S.D. N 3.86 3.03 72 524 568.46 1103.35 2.02 68 7.38 8.88 2.43 56 mmol/L 11.30 3.89 Creatinine μmol/L 83.20 Bun / Creatinine ratio ratio Lactose Dehydrogenase S.D. N 3.26 3.28 53 72 610.11 525.89 54 1.24 4 7.91 2.88 11 8.13 2.32 72 8.69 2.71 57 567 9.84 2.85 72 11.14 4.16 57 24.00 553 77.32 19.25 71 88.26 29.88 57 0.15 0.08 553 0.13 0.05 71 0.14 0.06 56 IU/L 1784.97 822.29 30 No data No data No data 2590.50 587.61 2 Amylase IU/L 893.31 490.28 74 655.60 269.49 5 753.60 388.75 15 Lipase IU/L 181.60 115.37 68 330.40 300.58 5 145.92 43.34 12 Tetra Iodothyronine nmol/L 57.70 16.40 128 56.48 21.64 25 65.22 27.92 13 Un-implanted mean Clinically abnormal mean Implanted mean Table 2.7. (cont.) Serum biochemistry values comparing clinically normal unimplanted and implanted and clinically abnormal Vancouver Island Marmots (Marmota vancouverensis). Bold indicates comparisons with statistical and potential clinical significance (a). S.D. N 4.61 5.35 455 IU/L 650.75 897.44 Cholesterol mmol/L 7.23 Glucose mmol/L Blood Urea Nitrogen Parameter Units Gamma Glutamyltransferase IU/L Creatinine Phosphokinase ELECTROLYTES AND ACID-BASE RENAL FUNCTION OTHER (a) Potential clinical significance was considered if the magnitude of the difference in the parameter comparison had practical relevance with respect to physiological or clinical state. 53 Table 2.8. Summary of significance levels in hematology parameters between specific treatments or groups of Vancouver Island Marmots (Marmota vancouverensis). Grey indicates statistical significance at α < 0.01 and black indicates that there is both Band neutrophils Lymphocytes Monocytes Eosinophils Basophils Red blood cells Hemoglobin Hematocrit Mean corpuscular volume Mean corpuscular hemoglobin Mean corpuscular hemoglobin concentration Red cell distribution width Platelet count Mean platelet volume Un-Implanted versus Implanted Un-implanted versus Nonrepresentative Segmented neutrophils Captive versus Wild Captive versus CaptiveRelease Wild versus CaptiveRelease White blood cells statistical significance at α < 0.01 and potential clinical significance (a). <0.01 0.90 0.23 <0.01 <0.01 0.08 0.14 0.48 0.55 1.00 <0.01 <0.01 0.76 <0.01 0.48 <0.01 <0.01 0.81 0.62 <0.01 0.34 0.87 0.45 0.61 0.96 1.00 <0.01 0.31 0.43 0.49 0.50 0.98 0.34 0.77 1.00 0.03 0.08 0.44 0.21 0.81 0.72 1.00 0.35 0.69 0.14 0.06 0.69 0.01 0.50 0.12 0.14 0.68 0.24 1.00 0.17 <0.01 <0.01 <0.01 0.25 0.02 0.02 0.03 <0.01 0.87 <0.01 <0.01 0.37 0.41 <0.01 0.84 <0.01 0.69 0.03 0.15 0.01 0.02 1.00 0.15 <0.01 0.21 (a) Potential clinical significance was considered if the magnitude of the difference in the parameter comparison had practical relevance with respect to physiological or clinical state. 53 54 Table 2.9. Summary of significance levels in serum biochemistry parameters between specific treatments or groups of Vancouver Island Marmots (Marmota vancouverensis). Grey indicates statistical significance at α < 0.01 and black indicates that there is Sodium / Potassium ratio Chloride Carbon Dioxide Anion Gap Calcium Phosphorous Calcium / Phosphorous ratio Calculated Osmolality Total Protein Albumin Globulin Albumin / Globulin ratio Total Bilirubin Alkaline Phosphatase Un-implanted versus Implanted Un-implanted versus Nonrepresentative Potassium Captive versus Wild Captive versus CaptiveRelease Wild versus CaptiveRelease Sodium both statistical significance at α < 0.01 and potential clinical significance (a). <0.01 <0.01 <0.01 0.01 <0.01 0.78 0.71 <0.01 <0.01 0.11 <0.01 <0.01 <0.01 0.78 0.17 0.05 <0.01 <0.01 <0.01 0.27 0.67 0.60 0.07 0.36 0.17 0.07 <0.01 <0.01 0.10 0.42 0.01 0.97 0.08 0.02 0.02 <0.01 0.31 0.71 0.64 0.17 0.72 0.17 0.56 0.31 0.06 <0.00 0.02 0.60 0.45 <0.01 0.82 0.96 0.96 0.96 0.99 0.82 0.95 0.98 0.84 0.79 0.92 0.95 0.93 0.92 0.55 0.83 0.97 0.97 0.97 0.93 0.99 0.85 0.96 0.98 0.94 0.94 1.00 0.98 0.88 0.94 (a) Potential clinical significance was considered if the magnitude of the difference in the parameter comparison had practical relevance with respect to physiological or clinical state. 54 55 Table 2.9. (continued) Summary of significance levels in serum biochemistry parameters between specific treatments or groups of Vancouver Island Marmots (Marmota vancouverensis). Grey indicates statistical significance at α < 0.01 and black indicates Alanine Aminotransferase Aspartate Aminotransferase Gamma Glutamyltransferase Creatinine Phosphokinase Cholesterol Glucose Blood Urea Nitrogen Creatinine Bun / Creatinine ratio Lactose Dehydrogenase Amylase Lipase Tetra Iodothyronine that there is both statistical significance at α < 0.01 and potential clinical significance (a). Captive versus Wild 0.02 0.19 0.88 0.20 <0.01 0.48 <0.01 <0.01 1.00 No data <0.01 0.48 <0.01 Captive versus Captive-Release 0.21 0.85 0.44 0.67 No data <0.00 0.07 0.02 0.73 No data No data No data No data Wild versus Captive-Release 0.26 0.06 0.21 0.42 No data 0.02 0.33 0.25 0.64 No data No data No data No data 0.91 0.94 0.96 0.97 0.99 0.83 0.89 0.93 0.93 No data 0.90 0.73 0.97 0.96 0.86 0.93 0.99 0.90 0.96 0.99 0.95 0.97 0.81 0.90 0.90 0.87 Un-implanted versus Implanted Un-implanted versus Nonrepresentative (a) Potential clinical significance was considered if the magnitude of the difference in the parameter comparison had practical relevance with respect to physiological or clinical state. 55 56 DISCUSSION In this project, reference intervals for 16 hematology and 30 serum biochemistry parameters were calculated from laboratory data derived from clinically healthy Vancouver Island Marmots over a 23-year period. In addition to providing baseline health data, these parameters were qualitatively compared to values that have been generated from other rodent species. The general characteristics of the VIM reference ranges appeared to be commensurate with values for other small to medium sized rodents. The hematology and serum biochemistry parameters also were used as a measure of comparison between different management VIM conditions (wild, captive and captive-release, un-implanted and implanted, clinically normal and abnormal). Eleven of the 46 parameters showed no clinical or statistical differences between any of the groups that were compared, including values which were derived from clinically abnormal animals. Twenty-eight of the 46 parameters showed evidence of statistically significant differences between certain groups, but the small magnitude and nature of these differences did not suggest physiological or clinical significance. Eight of the 46 parameters had biologically plausible and statistically significant differences between a portion of the compared groups. This included 5 leukogram parameters; white blood cells, segmented neutrophils, lymphocytes, monocytes, basophils, and 3 serum biochemistry parameters; total protein, albumin, and globulin. Potentially significant differences in leukogram and protein parameters occurred between the captive and free-ranging groups (both wild and captive-release). Also, leukogram parameters of clinically normal, un-implanted marmots were lower than the clinically-abnormal group. Although the blood samples from the clinically abnormal animals were collected under a range of 57 different scenarios, at least 9 were obtained from marmots with confirmed infections and elevated white blood cells and neutrophils. In addition to indicating inflammation or infection, white blood cells (including neutrophils, lymphocytes, monocytes, and basophils) and serum proteins also are indicators of general stress and immune status (Mellish et al. 2010; Barker & Boonstra, 2005). Although group characteristics for leukograms and serum proteins still fell within the established references ranges and the magnitude of differences between the groups were relatively small, these results suggest that leukogram and protein values may have the potential to differentiate the physiological or immunological status of healthy individuals in the different management groups. Although hematology and serum biochemistry parameters are commonly advocated as a tool for the evaluation and comparison of health in individuals and populations (Ruykys et al., 2012; Mellish et al., 2010; Masello & Quillfeldt, 2004), there are many factors in addition to health status which may have the potential to influence blood values and reference ranges. This includes intrinsic factors such as age, sex, and seasonality (Stannard et al., 2013; Barker & Boonstra, 2005) and extrinsic factors such as sample collection, storage and transport, and laboratory methods of analysis (Dimauro, et al., 2008; Low et al., 2006). The challenge of using this type of data is determining which parameters are restricted to the evaluation of individuals, which vary according to factors like age and sex, which vary due to vagaries associated with sample handling and laboratory analysis, and which represent legitimate differences between individuals or groups under different treatments. Although statistically significant differences often may be identified, it is also important to consider the magnitude of these differences and their clinical, physiological, or biological relevance or implications (Nakagawa & Cuthill, 2007). 58 In several instances, differences between groups could possibly be attributable to extrinsic factors. Analytical methodologies may have varied between laboratories (Low et al., 2006) and techniques may have altered over time. Due to the cost of analyses, it is also possible that institutions were somewhat selective in terms of which individuals they sampled, focusing on genetically important or older individuals. Pre-analytical factors, such as sample collection, handling and storage, may account for higher variation in results than analytical techniques (MacedaVeiga, et al., 2015). Although attempts were made to standardize the conditions under which blood was collected, stored, and transported, it is possible that some of the samples obtained from free-ranging marmots were artifactually compromised (Low et al. 2006). The measurement of lactose dehydrogenase, sodium, potassium, chloride, phosphorous, blood urea nitrogen, creatinine and glucose have all been shown to be influenced by temperature and storage interval (Monden et al. 2008; Reese et al. 2006). Under field conditions, some marmot samples may have been exposed to logistically imposed variations in ambient temperature and transport delays prior to laboratory analysis. This might represent a consideration in interpreting comparisons between free-ranging and captive marmots. It is important for ongoing species recovery and future decision-making to better understand if perturbations or artificial manipulation, including captive management and implant surgeries, are having an observable impact on the health of the species. In this project (i) the reference values for clinically healthy Vancouver Island Marmots are comparable to those of other related mammal species, (ii) the parameters of marmots released from captivity are comparable to their wild counterparts, (iii) the parameters of marmots that have been surgically implanted with abdominal radio-transmitters do not differ in any clinically significant way from healthy marmots without transmitters, (iv) healthy captive marmots 59 demonstrate higher, statistically significant leukogram and serum protein parameters, compared to healthy free-ranging individuals, which is possibly suggestive of increased chronic stress or subclinical health influences (Mellish et al. 2010; Barker & Boonstra, 2005), and finally, (v) white blood cell and protein metrics, in conjunction with other clinical data, may present a simple, efficient, and costeffective parameter for monitoring differences between management groups. Hematology and serum biochemistry have great utility for assessing health and identifying disease in individuals. Recognising abnormality or dysfunction in these parameters requires an appreciation of normal and this is facilitated by the establishment of baseline measurements. Although the utility of these measurements for assessing group or population effects are not as straightforward, monitoring these parameters over time may allow for the identification of increased individual dysfunction, for ongoing comparisons between the different management groups, and for continued surveillance of the marmots’ overall health status. Recognition of future alterations in hematology or serum biochemestry characteristics, in concert with other clinical data, may help to signal changes in health. This in turn may serve to identify emerging health risks that could influence the species’ overall health and recovery. 60 LITERATURE CITED Armitage, K. B. (2014). 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Environment influences morphology and development for in situ and ex situ populations of black-footed ferret (Mustela nigripes). Animal Conservation 8, 321-328. 64 CHAPTER 3 SURVEILLANCE OF VANCOUVER ISLAND MARMOT (MARMOTA VANCOUVERENSIS) HEALTH USING CLINICAL AND POST MORTEM EXAMINATIONS, AND FIELD MORTALITY DETERMINATION INTRODUCTION Guidelines produced by the International Union for Conservation of Nature (IUCN) on the management of ex-situ populations, reintroductions, and other conservation translocations, have identified health and disease monitoring as an essential component in an effective conservation program (IUCN, 2013; IUCN, 2002). Monitoring helps to identify ongoing or novell threats and allows for the adaptation of management, release, and surveillence regimes. The establishment of characteristic health parameters in captive, captive-release and wild populations delineates what is normal or expected under each treatment and how these differ between groups (Deem et al. 2001). Differences in parameters may reflect a response to disparate conditions or they may signal a potential health challenge associated with management or emerging stressors. Despite their widely acknowledged potential to influence success in conservation programs, the effects of animal health and disease are infrequently reported (Mathews et al. 2005). Monitoring health and disease in ex situ and in situ populations presents a significant challenge. Determining the spectrum of influence that captive conditions have on the health of an endangered species is timeconsuming and complicated and monitoring free-ranging populations is limited by the challenges associated with observing, examining, sampling and tracking individuals under field conditions. The health data obtained from these respective populations often is of disparate quality and this makes comparisons problematic. 65 Identifying potential health threats requires the organization and collation of data from a variety of sources. Grouping or clustering health data may have the potential to facilitate comparisons and allow timely detection of potential trends. Syndromic surveillance involves the systematic collection and evaluation of a wide range of health data from multiple sources to identify, characterize, and predict potential patterns, clusters, or syndromes as they are occurring. This allows for the detection of non-specific trends and for the tracking of specific syndromes using incomplete or imperfect data. This type of surveillance facilitates the filtering and efficient transfer of health or disease information so that risks can be identified and further investigated, and so that timely, informed decisions can be made. Although syndromic surveillance has been most commonly used to filter and identify syndromes in large, complex datasets, it may also present a conceptual framework for the detection of health and disease patterns in ex situ and in situ populations of endangered species, which may only be signaled by incomplete data from a relatively small number of individuals or events. This allows for the timely detection, comparison, investigation, and management of health risks before definitive diagnoses can be fully realized. The Vancouver Island Marmot (Marmota vancouverensis) is a critically endangered sciurid endemic to the mountains of central Vancouver Island (Swarth, 1912; Swarth, 1911). In the early to mid-1980s there were indications that the marmot’s population was stable or increasing, with approximately 300 to 350 individuals occupying at least 30 colony sites. However, in the late 1980s and throughout the 1990s, the Vancouver Island Marmot (VIM) demonstrated precipitous declines. By 1998 the wild population had dropped below 100 animals and by 2003 it was reduced to less than 30 individuals at 5 colonies, making it 66 Canada’s most endangered mammal (Jackson et al. 2015; Nagorsen, 2005; Janz et al. 2000). The impending threat of species extinction led to the initiation of an intensive captive breeding program in 1997. Since its inception, this program has involved the participation of three Canadian zoological institutions, the Toronto Zoo (TZ), the Calgary Zoo (CZ), and the Mountain View Conservation and Breeding Society in Langley, British Columbia (MV) and the construction of a dedicated marmot facility at Mount Washington on Vancouver Island, the Tony Barrett Mount Washington Marmot Recovery Centre (MRC). Although there has been significant recovery of the wild population, the VIM continues to be managed by an intensive program of captive breeding, reintroduction, and translocations. Further details on the history of the VIM recovery project are provided in Chapter 1. In situ and ex situ management has subjected the VIM to a unique and dynamic assemblage of natural and artificial influences. The ability to identify and track these influences with respect to marmot health is vital for effective long-term management and for vigilent recognition, ellucidation, and mitigation of potential risks that may jeoprodise the recovery of this species. The marmot recovery project has generated a great range of qualitative and quantitative, health-related data from both free-ranging and captive marmots. These have originated from a multitude of sources, and include clinical, pathological, morphological, physiological, demographic, epidemiological, and environmental data. Because of its disparate, individualistic, and heterogeneous nature, the utility of these data for elucidating marmot health patterns has not been fully realized. Clinical evaluations conducted by veterinarians, necropsies performed by veterinary pathologists, and properly vetted observations made by field staff monitoring free-ranging marmots through radio-telemetry, have the potential to 67 offer reliable and robust health-related data. Although this information has been typically used to evaluate individuals or singular events, it also has the potential to be extrapolated more broadly. In this chapter I use syndromic surveillence of disparate, historical data from physical examinations, post mortem examinations, and field evaluation of marmot mortalities, to identify possible trends and risk factors relating to the health of captive, captive-release and wild populations of VIM, and to evaluate the utility of this approach in informing future marmot health management and species recovery. MATERIALS AND METHODS Classification of marmot management groups Captive marmots were defined as any captive-born marmot originating from the TZ (Toronto, Ontario), the CZ’s Devonian Wildlife Conservation Centre (De Winton, Alberta), the MV (Langley, British Columbia) and the MRC (Mount Washington, British Columbia) or any wild-born marmot that was maintained at one of these facilities for more than a single, active season for the purpose of captive breeding. Four wild-born marmots that died during their initial 30-day quarantine period following capture were intended to be part of the captive breeding program and were defined as captive marmots even though they were not maintained beyond one active season. Wild-born marmots that were held for brief intervals at the MRC (periods of time that ranged from hours to weeks), and then re-released during the same active season, were classified as wild. These individuals were captured and held to simplify translocations and were never intended to be part of the captive breeding program. This latter group did not suffer any mortalities during their limited time in captivity. 68 From 1997 to 2016, a total of 639 individuals were maintained in captivity, including 61 wild-born marmots originally captured from the wild and 578 marmots (from 170 litters) born and weaned in captivity. This number does not include marmots from at least 11 unsuccessful, captive litters (6.1% of total litters) in which all pups failed to survive to weaning age (at approximately 28 to 30 days) or individual pups that were part of successfully weaned captive litters that failed to survive to weaning age. Captive-release marmots were defined as those animals released to the wild from captivity, including those originally born in captivity and any wild-born marmot that had been maintained in captivity for more than a single active season prior to release. From 2003 to 2016 a total of 490 captive marmots were released to the wild including 482 captive-born individuals (103 born and weaned at TZ, 116 at CZ, 98 at MV and 165 at MRC) and 8 wild-born individuals (time in captivity ranged from 394 to 4350 days, average = 2091 days). Two of these wild-born, captive-release marmots were recaptured and permanently returned to captivity. All 490 captive marmots that were released to the wild were maintained at the MRC for a minimum of 30 days prior to release, as a way of verifying their health and minimizing the potential for introduction of novel pathogens into the extant free-ranging populations. From 2003 to 2012, all 373 of the captive non-pups released to the wild spent at least one hibernation at the MRC. The average interval that these marmots spent at the MRC prior to release was 450 days (range 207 to 2271 days). From 2008 to 2011, 35 pups born at the MRC were released to the wild in their first summer (average age 112 days, range 93 to 123 days). From 2013 to 2016 a total of 82 captive marmots, originating from the other captive facilities, were released to the wild during the same year in which they arrived at the MRC. The average interval of time that these marmots spent at the MRC prior to release, was 69 58 days (range 42 to 77 days). Overall, 50.3% of the releases from captivity occurred in July, 27.8% in August, 14.0% in September, 7.4% in June, 0.4% in May, and 0.2% in April. Pre-conditioned marmots were defined as captive-release individuals that were translocated to a new site after successfully surviving at least one hibernation in the wild. This treatment primarily represented an attempt to mitigate the poor survival of captive-release marmots introduced directly into Strathcona Park. Captive marmots were released at Mount Washington, where survival was consistently high, and then subsequently recaptured and moved after at least one winter in the wild. In 2008 one captive-release individual was opportunistically relocated to Mount Cain after successfully surviving its first wild hibernation at Mount Washington and from 2012 to 2016, a total of 32 pre-conditioned marmots were trapped and translocated from Mount Washington to Strathcona Park. The interval between initial release from captivity and subsequent relocation ranged from 316 to 1042 days (average = 402 days). Between 2003 and 2009, four captive-release marmots that had moved into unsuitable habitat shortly after their initial release were recaptured and returned to their original release site or to a new site and were not considered to be pre-conditioned. Wild marmots were defined as any wild-born individual that had never been maintained in a captive facility or any wild-born marmot temporarily held at the MRC for less than a single, active season and then returned to the wild. Translocation marmots were wild marmots that were deliberately moved from one site to another. From 1996 to 2015, fourteen wild marmots were translocated to various sites in the Nanaimo Lakes region. From 2012 to 2016 a total of 58 wild marmots were translocated from Mount Washington to Strathcona Park. 70 Captive-release, pre-conditioned, wild, and translocation marmots were collectively defined as ‘free-ranging’. Marmot ages were categorized as young-of-the-year (pups or juveniles in their first summer), yearling (individuals in their second summer), two-year old (third summer), and adults (fourth or subsequent summers). The age of all captiveborn individuals was known with certainty. The age categories of wild-born marmots were known or extrapolated from previously described pelage characteristics (Bryant 1998) and relative size and appearance. In the field, age categories could be determined with relative certainty, except in the case of some individuals classified as two year-olds, that exhibited variable characteristics which overlapped those of yearlings and adults. Monitoring of free-ranging marmots Free-ranging marmots were monitored in the field with surgically implanted, intra-abdominal VHF (very high frequency) radio-transmitters. Internal transmitters were used because the dramatic seasonal mass changes that individuals demonstrate during hibernation made conventional neck collars impractical. From 1992 to 2016 a total of 898 implant surgeries were performed on VIM including 96 replacement surgeries. Techniques for surgical implantation of intraperitoneal transmitters have been previously described in a range of wild species (Van Vuren, 1989; Ranheim et al. 2004; Soto-Azat et al. 2008) and the specific details concerning transmitters and handling, anesthetic and surgery techniques used on this project are described in more detail in Appendix D, E and F. Because implantation required a surgical procedure and an adequate capacity for convalescence, marmots were not implanted close to hibernation (i.e. early in the field season following emergence, when body condition and food resources were low and when female marmots might be pregnant or late in the field season close to immergence when the marmots’ 71 metabolic rate was starting to decline and healing capacity might be reduced). Most surgeries were performed between the middle of June and the end of August. During the course of this project, temperature-responsive transmitters from Custom Telemetry ® (Watkinsville, Georgia), Telonics ® (Mesa, Arizona), Advanced Telemetry Systems ® (Isanti, Minnesota) and Holohil Systems Limited ® (Carp, Ontario) were used. The pulse rate and temperature response of the transmitters was configured to maximize functionality and prolong battery life. A reduced pulse rate associated with lowered body temperature helped to prolong battery life during hibernation and during the active season a low transmitter pulse rate was used to signal potential marmot mortalities. Four hundred and eighty-four of the 490 captive-release marmots were surgically implanted with an abdominal radio transmitter prior to release and then afforded a period of convalescence in captivity. Intervals between captive surgery and release ranged from 11 to 93 days (average = 28 days). Three implanted marmots were held until the following year due to transient problems or injuries that precluded same-year release and one individual died prior to release due to transmitter complications. One marmot was recaptured and surgically implanted 50 days after being initially released without a transmitter and 32 individuals were later recaptured and had their transmitters replaced. One captive-release marmot was recaptured for a second transmitter replacement. From 1992 to 2016 a total of 379 implant surgeries were performed on wild marmots including 63 replacement surgeries. Wild marmots being intentionally prepared for translocation were surgically implanted with radio-transmitters and then allowed to convalesce at their familiar colony sites for a period which ranged from 21 to 37 days (average = 30 days). In five instances, wild marmots were not recaptured and translocated until the year following their surgery. Four marmots 72 that were captured at aberrant locations including Bamfield, Nanaimo (2), and Nanoose on Vancouver Island were surgically implanted and translocated to suitable habitat within days of initial capture. All four marmots survived for at least 350 days (range 350 to 1511 days), indicating that these marmots were not adversely affected by prompt relocation following surgery. Physical Examinations Comprehensive physical examinations were conducted on both captive and free-ranging VIM. These examinations were conducted by a veterinarian and involved an evaluation of the cardiovascular, respiratory, musculoskeletal, nervous, integumentary, and urogenital systems, and an assessment of the marmot’s body condition and weight. Individuals with previously implanted radio-transmitters were routinely palpated to ensure that the units were mobile or free-floating within the abdominal cavity. Marmots that did not display identifiable physical abnormalities were classified as clinically normal or healthy at the time of examination. Prior to physical examination, most marmots were immobilized with an intramuscular injection of ketamine hydrochloride (10 mg/kg) and midazolam hydrochloride (0.25 mg/kg), and then maintained on inhaled isoflurane. In some instances, marmots were mask induced with isoflurane without receiving any injectable immobilization agents. Physical examinations of VIM were conducted under the following conditions: 1) Captive marmots: i) In addition to examinations initiated during quarantine (total = 55) or in response to specific health concerns (61), all captive marmots maintained at the TZ, CZ, MV and MRC received routine physical examinations on an annual or biennial basis. These examinations were conducted late in the 73 active season in anticipation of hibernation or prior to transfer to another captive facility. ii) Captive-release, pre-release - 485 of the 490 marmots released from captivity were surgically implanted with radio-transmitters and were given pre-operative examinations at MRC to confirm their suitability for surgery. There was also a pre-release examination which followed the post-operative, convalescence period. This second examination occurred on the day of release or on the day that preceded it. The 5 un-implanted marmots also received pre-release exams. 2) Free-ranging marmots: i) Captive-release, post-release - marmots were examined if they were recaptured for transmitter replacement surgery. ii) Captive-release, pre-conditioned - following recapture, these marmots were typically held for a short interval (hours to weeks) at the MRC and then examined on the day of their re-release to a new site or on the day that preceded it. iii) Wild marmots receiving transmitters were given pre-operative examinations to confirm their suitability for surgery. iv) Wild marmots also received examinations if they were re-trapped for transmitter replacement surgery. v) Thirty-eight out of 61 wild marmots were examined shortly after capture but prior to being transferred to the captive breeding program. vi) Wild marmots, translocation - these marmots were recaptured after an in situ convalescent period and typically held for a short interval (hours to days) at the MRC. These marmots were re-examined on the day of their translocation or on the day that preceded it. 74 Post Mortem Examination / Causes of mortality All marmots found dead in the wild or in captivity underwent a standardized post mortem examination. Necropsies were performed by pathologists at the Province of British Columbia’s Animal Health Centre (Abbotsford, BC), the Province of British Columbia’s Wildlife Branch (Victoria, British Columbia), the Calgary Zoo (Calgary, Alberta) and the Ontario Veterinary College (University of Guelph, Guelph, Ontario). Wherever possible, the same pathologists were recruited to perform or review post mortem examinations, so that these individuals developed increased experience in recognizing, comparing and interpreting lesions and abnormalities, and were better able to identify potential patterns. The attending pathologist had the discretion to determine the thoroughness of the post mortem examination and to perform ancillary diagnostic procedures, as they felt necessary. The thoroughness of a post mortem examination also was determined by the circumstances under which a marmot was recovered. Captive VIM that died or were euthanized during the active season were examined within hours or days of death. This facilitated gross, histological and microbiological examination. Captive marmots that died during the hibernation period were most commonly recovered during routine nest-box checks, conducted at weekly to monthly intervals, and their carcasses demonstrated varying degrees of autolysis. Deterioration of the carcass was somewhat delayed by the cooler ambient temperatures (5 to 7° C) that characterized hibernation management. Typically, the tissues of these marmots maintained sufficient integrity for a macroscopic post mortem examination, but the potential for histological and microbiological examination was greatly reduced. Free-ranging VIM could not be monitored with the same day-to-day intensity as their captive counterparts, and this complicated the field recovery of intact, dead marmots. Although field workers occasionally encountered the remains of 75 unidentified marmots of unknown origin, most of the documented mortalities in free-ranging marmots involved individuals that were previously implanted with radio-transmitters and located by telemetry. Hibernation mortalities were identified in those marmots that failed to emerge from their burrow in the spring and whose transmitter remained underground on a slow pulse rate. If there were no indications as to cause of death following site investigation or if a slow transmitter pulse was detected remotely by aerial or ground telemetry, the cause of death was listed as “unknown”. The interval between death and recovery of free-ranging marmot remains was quite variable and ranged from days to years. The recovery of free-ranging marmots which had died during hibernation occurred on only two occasions and necessitated digging the dead marmots out of their hibernaculum. Decomposition of these recovered carcasses was probably delayed by the cooler and more stable, ambient temperatures that characterize natural burrows. The integrity of remains from dead, free-ranging marmots also could be influenced by consumption and disruption by predators or scavengers, moisture (rain or snow), sunlight and ambient temperatures. In most instances, the death of a free-ranging marmot resulted in the recovery of incomplete remains, which ranged from an isolated transmitter to a partial body. In the absence of a complete carcass, the proximate causes of a marmot mortality were determined by site investigation and the characteristics of the marmot remains. Classification of predator mortalities VIM are susceptible to a suite of predators (cougars, wolves, eagles, and bears), and each species typically left a characteristic presentation of the remains following a predation event. 76 Cougars (Puma concolor) are solitary hunters. Once a cougar successfully killed a non-juvenile marmot, it typically delayed feeding and relocated the carcass to a sheltered spot under the cover of dense vegetation (Naughton, 2012). Often these sites were situated well away from locations typically frequented by marmots. Cougars fastidiously prepared the marmot’s body prior to consumption. They used their incisors to barber or pluck the marmot’s fur, leaving an encircling perimeter or matt of hair. They typically removed and rejected the stomach and gastrointestinal tract of their prey (Wild, 2013) and where applicable, the implanted abdominal transmitter. Cougars also disarticulated the carcass during feeding and in many instances, they left the more robust bones (skull, pelvis, and long bones) and the poorly fleshed portions of the body (feet and tail). Grey Wolves (Canis lupus) are pack hunters and display competitive behavior following a predation event. They typically consumed marmots at the location of the kill, and ingested most of the carcass, including the bones, gastrointestinal tract, and skin. They would only leave small remnants such as the upper incisors mounted in a portion of the premaxillary bone, and a portion of the distal tail. Wolves would not consume abdominal transmitters, but they would frequently leave bite marks on the surface of units, particularly those that were encapsulated with wax. In addition, wolves often showed what appeared to be a gastro-colic response, and tell-tale feces often were found in close proximity to their marmot kills. The preferred prey size for Golden Eagles (Aquila chrysaetos) is 0.5 to 4.0 kilograms (Watson, 1997) and most free-ranging VIM fall within this range. In many instances, Golden Eagle predation on full-size marmots left indications of an initial struggle, characterized by disturbance of low-lying vegetation, and feather and body tracks on the ground or in the snow. The eagle’s presence often was evidenced by down and contour feathers, and by large splashes of urates. A female Golden Eagle 77 has difficulty flying with a prey item that matches her own body weight (approximately 6.0 kilograms), and under most conditions, she can only manage to carry about half of this amount (Watson, 1997). Therefore, Golden Eagles often began consuming marmots at the kill site, leaving partial remains, including the skeleton and some viscera, surrounded by irregular patches of plucked hair. In a few instances, field workers flushed Golden Eagles off their marmot kills. On three occasions Golden Eagles were observed carrying small marmots (pups or yearlings) away from colony sites (Bryant, 1998) and at least one marmot radio-transmitter was used to locate a Golden Eagle nest (J. MacDermott, personal communication). Black Bears (Ursus americanus) were random, opportunistic predators of marmots and this was reflected in the apparently haphazard nature of carcass consumption. Bears would typically consume most of the bones and soft tissues and leave behind a twisted hide with irregular remnants of the subcutaneous fat and panniculus muscles, which was coated with detritus such as leaves, dirt, and broken twigs. In cases of bear predation there was also proximal evidence of the bear’s presence, such as piles of feces or a bedding area. Because of their small size, most marmot pups were probably carried away or consumed in their entirety after being killed. Implanted pups left negligible remains except for occasional clumps of hair, skin, and their transmitter. 78 RESULTS Physical Examination From August 1992 to September 2016, a total of 3,174 veterinary examinations were conducted on captive and free-ranging VIM. This includes examination of 632 captive individuals and 353 wild individuals. Accounting for marmots that overlapped the two populations, a total of 944 individual marmots were examined. The circumstances of these examinations are summarized in Table 3.1 and the ages of wild and captive marmots are compared in Figure 3.1. On average, captive marmots were examined more frequently than wild marmots (an average of 4.1 examinations per captive marmot compared to 1.4 examinations per wild marmot). Physical evaluation of captive marmots resulted in the identification of a number of health events or clusters that were not seen in freeranging marmots (Table 3.2). Although seven weaned pups died before receiving physical examinations, all of the remaining 571 captive-born pups were evaluated prior to their first hibernation. This compares to 63 wild pups who were either examined before being transferred into captivity (27) or prior to implant surgery (36). Captive-born pups exhibited a number of congenital disorders that were not identified in wild pups including a patent foramen ovale, bilateral cataracts, dental malocclusion, and bilateral aplasia of the hind feet. One wild-born pup that was brought into captivity possessed an atrial septal defect and his full sibling (born after the wild-born parents had been taken into captivity) exhibited stunting, microopthalmia, and scoliosis. The greater longevity of captive marmots allowed for observation and physical examination of many older marmots, compared to their wild counterparts (Figure 3.1) and this may have allowed for the identification of additional age-related health events or syndromes within the captive population. Although cardiovascular disease and neoplasia often were diagnosed at the time of 79 necropsy following acute death in captive marmots, some of these cases were preceeded by observable clinical signs. Marmots with chronic heart disease exhibited murmurs or weight loss that were detected clinically and initial signs of neoplasia included emaciation, depression, inappetance, facial assymetry, epistaxis, and unilateral exopthlamos. Two mature, captive males exhibited acute hind end paralysis that resulted from degeneration and prolapse of their invertebral discs. Cardiovascular disease, neoplasia and disc degeneration were not identified in any of the free-ranging marmots that were given physical examinations. One condition that was exclusive to wild-born marmots was a syndrome of chronic, generalized, perifollicular dermatitis that was widespread in the original, extant colony at Mount Washington and was attributed to an unclassified, intrafollicular mite (Janz et al., 2000). This condition persisted in those individuals that were taken into the captive program, but it did not visibly affect their progeny or cagemates. 80 Table 3.1. Circumstances of 3,174 physical examinations conducted on captive and free-ranging Vancouver Island Marmots (Marmota vancouverensis). Free-ranging Captive (2622) Captive-release (67) Wild (485) Annual / biennial exam Pre-surgical (a) Pre-release exam (b) Other (c) Pre-surgical (a) (d) Implant replacement Translocation (pre-conditioned) Pre-surgical (a) Implant replacement Translocation Captivity (e) Other (f) 1513 486 507 116 1 33 33 316 63 61 38 6 a) Examination prior to surgical implantation of abdominal radio-transmitter b) Some marmots had more than one pre-release exam due to delays in their release or concerns about transient injuries. One implanted marmot died prior to release due to complications associated with its transmitter c) Examinations of wild-born marmots conducted in the initial captive quarantine period or exams initiated in response to specific health concerns d) One individual was surgically implanted 50 days after being released from captivity without a transmitter e) Physical examinations conducted on captured wild-born marmots prior to being placed in the captive program f) Miscellaneous examinations associated with ear-tag replacement or misidentified marmots that already had pre-existing transmitters 80 5 81 Figure 3.1. Age comparison of 3,107 physical examinations conducted on wild and captive Vancouver Island Marmots (Marmota vancouverensis). 900 No. of examinations 800 700 Total wild (485) 600 Total captive (2622) 500 400 300 200 100 0 0 1 2 3 4 5 6 7 8 9 Age-class (years) 10 11 12 13 14 81 5 82 Post Mortem Examination / Causes of Mortality Mortality in Captive Marmots: As of January 2017, 109 marmot deaths had occurred in captivity (49 wild-born and 60 captive-born). A post mortem examination was performed on 106 or 97.22% of the 109 captive mortalities. Examinations were not performed on two recently weaned pups whose bodies were not recovered. The body of a third pup that died from known conspecific trauma, was too badly mutilated for a necropsy. In one adult post mortem, advanced autolysis of the carcass (recovered during hibernation) precluded a diagnosis. A specific cause of death was identified in 106 of the mortalities, and the diagnostic categories are summarized in table 3.1. The sex-age distribution of the mortalities is summarized in figure 3.2. The average male age was 6.37 years (range 0.1 to 11.5) and the average female age was 8.17 years (range 0.1 to 14.6). Of the 78 marmots that survived to adulthood at 3 years of age, the average age for males was 8.9 years (n = 46) and the average age for females was 10.4 years (n = 32). Cardiovascular disease accounted for the highest number of captive marmot mortalities, and included 16 cases of dilatative or dilated cardiomyopathy, 6 cases of myocarditis, 1 case of endocardiosis, 1 case of cerebral hemorrhage, 1 case of atherosclerosis, 4 cases in which the pathologist diagnosed non-specific heart disease, and 3 cases which were described as congestive heart failure. One five-year old, captive-born male suffered cardiac arrest during anesthesia and a captive-born male died acutely of myocardial fibrosis at 1.4 years of age. Including two additional congenital cases (atrial septal defect, patent foramen ovale), 27 captive males died from cardiovascular disease compared to 9 females. Twenty-four males died of acquired cardiovascular disease in adulthood and their average age was 9.5 years (range 5 to 11.5). In the 9 females, the average age was 11.8 years (range 10.3 to 13.1). 83 Neoplasia occurred in combination with 5 of the 34 cardiovascular cases (3 males, 2 females) and was identified in 21 additional mortalities (8 males, 13 females). The anatomical categorization of these cases is presented in Table 3.4. The average age for the 11 males with neoplasia was 9.0 years (range 6.4 to 11.5) and for the 15 females it was 10.4 years (range 8.2 to 14.6). A total of 25 (22.9%) of the captive deaths were attributed to infections or inflammation. This include 6 cases with multisystemic involvement, 6 pulmonary, 5 hepatic, 2 pancreatic, 2 gastrointestinal, 2 neurological, 1 urogenital, and 1 integumentary. There were two instances in which captive pups exhibited severe neurological symptoms and were ultimately euthanized. At post mortem, both pups were diagnosed with meningoencephalitis. The etiological agent in one case was a protozoan, possibly Sarcocystis neurona and in the other it was the microsporidia, Encephalitozoon cuniculi. Of the 109 marmot mortalities that occurred in captivity, 17 (15.6%) were attributed to management or captive conditions. Six weaned pups were fatally attacked by older, non-related marmots after they escaped through the mesh of their natal enclosures. Four of the 61 wild-caught marmots died in quarantine during the intial transition to captive conditions. Three of these deaths were attributed to bacterial infections and one resulted from a cecal perforation arising from the microbial alterations associated with a new, captive diet. One marmot died from trauma associated with a fall in its enclosure and one was euthanized following elbow trauma of unknown origin. A captive pup scheduled for release experienced an extreme, chronic inflammatory reaction to its implanted transmitter, resulting in significant encapsulation and visceral adhesions which could not be surgically resolved. Two captive marmots died of hypothermia after being exposed to hibernation temperatures of -7° C while occupying an outdoor nest-box. 84 From the winter of 1997/98 to the winter of 2015/16 there were a total of 1651 individual marmot hibernations in captivity with 26 mortalities, indicating a success rate of 98.4%. Over the course of these 19 winters there were 547 pup hibernations (first winter) and 264 yearling hibernations (second winter) in captivity, with only 1 pup mortality. There was an additional adult mortality in December 2016, bringing the total number of captive hibernation mortalities to 27. Underlying pathology was identified in 24 of these hibernation deaths. Mortality occurring during captive hibernation most typically involved older marmots with identifiable health problems. This included 12 cases of cardiovascular disease, 4 cases of neoplasia, and 1 case of cardiovascular disease in combination with neoplasia. Three older marmots also succumbed to infections or inflammation during hibernation. The hibernation deaths of 4 younger captive marmots also were attributed to infection or inflammation. At least some of these mortalities appear to be bacterial or fungal in origin. However, most hibernation mortalities were not immediately recovered, and therefore, microbiology results should be interpreted with caution (de With et al. 1999). 85 Table 3.2. Clinical conditions initially identified from 3,174 Vancouver Island Marmot (Marmota vancouverensis) physical examinations. A total of 944 marmots were examined, including 632 captive individuals and 353 wild individuals. Sixtytwo marmots were examined following release from captivity. Category Diagnosis Occurrence in free-ranging marmots (wild & captive release) Occurrence in captive marmots Comments cardiovascular persistent tachycardia 0 1 heart rate consistently above 300 bpm cardiovascular acquired cardiac disease 0 28+ cardiac murmur congenital / early onset atrial septal defect 0 1 identified in wild-caught, captive yearling exhibiting un-thriftiness congenital / early onset patent foramen ovale 0 1 cardiac murmur congenital / early onset dental malocclusions 0 2 overgrowth of upper and lower incisors (both cases were litter-mates) congenital / early onset post weaning un-thriftiness 2 wild 2 significantly smaller than litter-mates congenital / early onset unilateral anopthalmis / scoliosis / stunting 0 1 captive-born marmot which was the progeny of wild-caught parents congenital / early onset hind foot aplasia 0 1 congenital / early onset bilateral, congenital cataracts 0 2 congenital / early onset unilateral, narrowed palpebral fissure 0 1 iatrogenic / management transmitter reaction 0 1 large abdominal mass infectious / inflammatory menigoencephalomyelitis 0 2 depression, ataxia infectious / inflammatory facial abscess 7+ unilateral facial swelling hibernation post hibernation emaciation 0 extreme emaciation and depression in captive-release marmot miscellaneous abdominal hernia 0 1 palpation of fluctuant peri-abdominal mass miscellaneous degeneration and herniation of intervertebral disc(s) 0 2 acute hind-end paralysis miscellaneous paraphimosis 0 1 possibly associated with neurological symptoms neoplasia facial neoplasia 0 7 epistaxis, facial assymetry, unilateral exopthalmus neoplasia other neoplasia 0 3 emaciation parasitic mites 25 wild 8 parasitic cutaneous myiasis 0 1 1? wild 1 captiverelease poor hair coat and hair loss, only occurred in wild-born marmots originally from Mount Washington bot fly larvae infesting skin of ventral abdomen 86 Table 3.2. (cont.) Clinical conditions initially identified from 3,174 Vancouver Island Marmot (Marmota vancouverensis) physical examinations. A total of 944 marmots were examined, including 632 captive individuals and 353 wild individuals. Sixty-two marmots were examined following release from captivity. Diagnosis Occurrence in free-ranging marmots (wild & captive release) Occurrence in captive marmots Comments traumatic unilateral / bilateral fractures of incisors 0 2 impaired regrowth of incisors following trauma traumatic fracture of carpal bones 1 wild 0 lameness traumatic chronic fracture of right olecranon 0 1 lameness traumatic head trauma 0 1 head tilt traumatic? resorption of head and neck of femur 0 1 lameness traumatic? unilateral, corneal opacity 1 wild 1 Category 87 Table 3.3. Categories of mortality observed in 109 captive Vancouver Island Marmots (Marmota vancouverensis), 1997 to 2016. Diagnostic category No. Cases % cardiovascular 29 26.6 infectious / inflammation 25 22.9 neoplasia 21 19.2 iatrogenic / management (a) 13 11.9 congenital / early onset (b) 6 5.5 cardiovascular & neoplasia 5 4.6 quarantine (a) 4 3.7 unknown 3 2.8 intervertebral disc degeneration 2 1.8 mesenteric torsion 1 0.9 (a) Iatrogenic / management was defined as any mortality directly related to the marmots being manipulated or confined in captivity. This included conspecific trauma, falls in enclosures, inappropriate hibernation temperatures and transmitter reactions. Death during the quarantine period was also attributed to captive confinement. (b) Early onset was defined as any condition diagnosed at the time of first examination either ante-mortem or post-mortem. 88 Figure 3.2. Sex-age distribution of 109 mortalities in captive, weaned Vancouver Island Marmots (Marmota vancouverensis), 1997 to 2016. 16 No. of cases 14 12 Males (61) 10 Females (42) 8 Unknown (6) 6 4 2 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Age class (years) Figure 3.3. Sex-age distribution of 518 mortalities in free-ranging Vancouver Island Marmots (Marmota vancouverensis), 1992 to 2016. 80 70 Males (273) No. of cases 60 Females (244) 50 Unknown (1) 40 30 20 10 0 0 1 2 3 4 5 6 7 8 Age class (years) 9 10 11 12 13 14 89 Table 3.4. Anatomical description of neoplasia in 26 captive Vancouver Island Marmots (Marmota vancouverensis) 1997 to 2016. Anatomical description No. Cases % pulmonary 6 23.1 hepatic 6 23.1 facial - oral 3 11.5 facial - periorbital 3 11.5 fascial - nasal 1 3.8 splenic 2 7.7 abdominal 1 3.8 adrenal 1 3.8 mammary 1 3.8 uterus 1 3.8 multi-systemic 1 3.8 90 Mortality in Free-ranging Marmots: From 1992 to the completion of the 2016 field season, a total of 533 confirmed mortality records were generated from free-ranging marmots. This included records for 183 wild marmots (including 44 translocated marmots) and 350 captive-release marmots (including 20 pre-conditioned marmots). Implanted marmots accounted for 515 (96.6%) of the mortality records. There were also 15 records in which field workers encountered the remains of unidentified marmots. In 5 of these cases, old or desiccated marmot remains (mostly small numbers of disarticulated bones and clumps of hair) were found in proximity to burrow entrances and were presumably pushed out of the burrow by other marmots. Although it is probably reasonable to assume that these marmots died underground, the timing of their deaths could not be reliably determined, and therefore the cause of death in these marmots was categorized as ‘unknown’. Causes of mortality of free-ranging marmots are summarized in table 3.5 and the sex-age distribution of these mortalities is summarized in Figure 3.3. The average age of males was 3.28 years (range 0.2 to 9.5) and the average female age was 3.57 years (range 0.2 to 13.1). Wild marmots From 1992 to 2016 a total of 379 implant surgeries were performed on wild marmots, including 63 replacement surgeries (total = 316 individuals). Mortality was confirmed in 167 or 52.8% of these implanted wild marmots. Only 7 (2.1%) of these telemetered marmots (4 wild and 3 translocated) were recovered in a condition which was suitable for a post mortem examination. The necropsy results for these 7 marmots (and one un-telemetered individual) are presented in Table 3.7. Four of these 7 wild marmots died following manipulation, including one individual which died from acute hyperthermia following handling and field surgery in 1992, and three translocated individuals which were recovered from their 91 hibernaculum in the spring of 1997 after a failed translocation attempt. A fourth marmot mortality from this same group could not be retrieved. These translocated marmots died during hibernation despite exhibiting good body stores and a definitive cause of death was not identified despite intensive investigation (de With et al. 1999). In addition to these four related mortalities, there was one other record of a translocated marmot that failed to emerge in the spring and its transmitter remained underground on a slow pulse. The intact carcass of a wild, telemetered marmot was recovered in May 2005 and post-mortem examination indicated that although it had been actively feeding aboveground (as evidenced by ingesta in its gastrointestinal tract) it had probably died of post-emergence emaciation. In 7 other instances, wild (non-translocated) telemetered marmots failed to emerge from their burrows in the spring, and their transmitters remained underground on a slow pulse rate. Only 1 of these 8 hibernation mortalities involving wild marmots occurred in the winter that followed implant surgery, with the remainder dying during subsequent winters. Captive-release From 2003 to 2016 a total of 490 captive marmots were released to the wild (annual numbers summarized in Figure 1.2 and Appendix A), including 482 captiveborn individuals and 8 wild-born individuals. A total of 485 of the captive-release marmots were surgically implanted with an abdominal radio transmitter for post release tracking and 33 of these individuals were later recaptured and had their transmitters replaced. Two of the 490 released marmots (one implanted, one not implanted) were recaptured and permanently returned to the captive breeding program. Mortality was confirmed in 349 (72.1%) of the 484 marmots fitted with radio transmitters that remained in the wild. 92 Only 22 or 4.55% of the 484 telemetered marmots were recovered in a condition that was suitably intact for a post mortem examination. In addition, a single un-telemetered, post-release marmot was recovered after drowning in a reservoir. No pre-conditioned marmots were recovered for necropsy. The necropsy results for these 23 intact, post-release marmots are presented in Table 3.8. Ten of the 23 post-release marmots presented for necropsy were diagnosed with post-emergent emaciation as the proximate cause of death. These marmots were discovered in May or early June. Eight of these marmots were found aboveground and two were dug out of a shallow hibernaculum. Telemetry on these latter two marmots indicated that their body temperatures were still cycling into periods of euthermy late into the spring before they died in their burrow. Another post-emergent marmot was still alive when it was recovered by field staff but perished while being transported into care. Extreme weight loss and loss of fat stores in a post-emergent, adult male may have resulted in an abdominal radio-transmitter becoming lodged in its pelvic canal, resulting in a fatal impaction of the gastrointestinal tract. At least one of the recently emerged marmots was found dead at the hibernaculum entrance, and it is possible that burrowing out through deep or frozen snow placed an additional burden on the marmot’s limited energy stores. In addition to the 10 cases where an intact carcass was recovered, there were 75 additional records (70 captive-release, 5 pre-conditioned) where post-release marmots died during hibernation. In these cases, a marmot failed to emerge from hibernation and its radio-transmitter continued to transmit a slow-pulse (i.e. low temperature) signal from the hibernaculum. Two marmots appeared to have emerged from hibernation and then died in their burrows shortly afterward. In at least two instances, a whole group of marmots failed to emerge from hibernation, and in at least two others, an individual marmot survived, whereas its burrow- 93 mates did not. Of the 80 hibernation mortalities documented in post-release marmots (excluding pre-conditioned marmots), 76 occurred in the first winter following release from captivity and 4 died during subsequent winters. Death during hibernation accounted for 44.4% of the captive-release mortalities for which there was an identified cause (N = 171), compared to 9.5% for unmanipulated (nontranslocated), wild marmots (N = 84). Predation accounted for 166 or 31.1% of the 533 mortalities in free-ranging marmots (Table 3.6). Mortalities attributed to predators are summarized according to year in Figure 3.4. 94 Table 3.5. Categories of mortality for wild, translocated, captive-release and preconditioned Vancouver Island Marmots (Marmota vancouverensis) including post mortem reports and mortality records, N = 533. Wild (n = 139) No. Cases % Predation 73 52.5 Hibernation 8 5.8 Other 3 2.2 55 (a) 39.6 No. Cases % Predation 5 11.4 Hibernation 5 11.4 Other 0 0.0 Unknown 34 77.3 No. Cases % Predation 86 26.1 Hibernation 80 24.2 Other 5 1.5 Unknown 159 48.2 No. Cases % Predation 2 10.0 Hibernation 5 25.0 Other 0 0.0 Unknown 13 65.0 Unknown Translocated (n = 44) Captive-release (n = 330) Pre-conditioned (n = 20) (a) This includes 16 records involving the remains of unidentified wild marmots encountered by field staff. 95 Table 3.6. Predator mortality for wild, translocated, captive-release and preconditioned Vancouver Island Marmots (Marmota vancouverensis) from 1992 to 2016 (total = 166). Wild (n = 73) Translocated (n = 5) Captive-release (n = 86) Pre-conditioned (n = 2) Total Cougar Golden Eagle Grey Wolf Black Bear Avian (a) Dog Unknown 38 8 9 0 1 1 16 0 1 2 0 0 0 2 38 25 8 1 0 0 14 0 1 0 1 0 0 0 76 35 19 2 1 1 32 (a) Marmot pup killed by avian predator, possibly Northern Goshawk (Accipiter gentilis) 96 Figure 3.4. Yearly predation mortalities of free-ranging Vancouver Island Marmots (Marmota vancouverensis) from 1992 to 2016 (total = 166). 25 Dog Undetermined predator 20 Number of mortalities Pup killed by avian predator (possibly Northern Goshawk) Black Bear Grey Wolf 15 Golden Eagle Cougar 10 5 0 1992 1994 1996 1998 2000 2002 2004 2006 2008 2010 2012 2014 2016 Year 96 5 97 Table 3.7. Post mortem diagnosis in 8 (7 telemetered, 1 non-telemetered) wild Vancouver Island Marmots (Marmota vancouverensis) 1992 – 2016. Diagnostic category hibernation hibernation No. Cases Comments group of 4 died in hibernaculum after failed translocation, 3 carcasses recovered (2 telemetered, 1 un-telemetered), etiology not determined post-emergent emaciation found aboveground 3 1 iatrogenic hyperthermia from handling & surgery 1 infectious hepatic abscess in a ten-year old female 1 predation suspect domestic dog 1 unknown post mortem inconclusive 1 Table 3.8 Post mortem diagnosis in 23 (22 telemetered, 1 non-telemetered) captiverelease Vancouver Island Marmots (Marmota vancouverensis) 2003 – 2016. Diagnostic category Comments No. Cases % post-emergent emaciation, found aboveground 8 34.8 group of 2 recovered in shallow hibernaculum 2 8.7 Golden Eagle 3 13.0 Cougar 1 4.3 drowning fell into reservoir 2 8.7 trauma fall from cliff 1 4.3 suspected trauma lesions suggested traumatic injury 1 4.3 iatrogenic transmitter impaction in pelvis 1 4.3 unknown post mortem inconclusive 3 13.0 hibernation (total =10) predation (4) 98 DISCUSSION The Vancouver Island Marmot recovery project has collected extensive information on both free-ranging and captive marmots, and from these data it is apparent that captive, wild and captive-release marmots are subjected to disparate assemblages of health determinants and exhibit different spectrums of health outcomes. In addition, there are significant differences in the capacity to collect health data and conduct surveillance on these populations. In captivity, VIM can be observed on a regular basis, and any suspected abnormalities in behaviour, appetite, excretion, or physical appearance (body condition, symmetry, posture, locomotion, hair coat, etc.) can be promptly recognized, evaluated, and monitored. In addition, captive marmots were routinely examined on an annual or biennial basis. Ninety-six per cent of the captive mortalities were efficiently identified and extensively investigated. Intensive observation, clinical evaluation, and post mortem examination of captive marmots resulted in the description of a number of health conditions. Although many of these conditions would present grossly observable signs, most were not identified in the numerous physical examinations that were conducted on free-ranging VIM (Appendix G). However, it is important to note that physical examinations were only conducted on those free-ranging individuals that could be successfully livetrapped and this may represent an important distinction in sampling between the captive and free-ranging populations. It is always possible that the health of some free-ranging marmots may have been compromised, and that the adaptive behavioral responses associated with “sickness behavior” precluded these marmots from being active and trappable above-ground (Hart, 1988). Predation, scavenging, ambient conditions, and delays in recovery reduced the effectiveness of mortality investigation in free-ranging marmots. Site accessibility also represented a 99 significant challenge in determining the occurrence, timing and causes of mortality in the field, and this varied greatly between locations. It is possible that the small number of intact carcasses that were recovered in the field for post mortem examination (31) also reflected the low number of non-predatory deaths that occur in the free-ranging population. One of the original objectives of the captive program was to establish a safeguard population, so that marmots could be maintained under controlled conditions without being exposed to the multiple threats that exist in the wild. However, captivity is not entirely benign and artificial conditions appear to have imposed a new set of risk factors for Vancouver Island Marmots. Cardiovascular disease was quite prevelent in captive male marmots and its onset was earlier than in females. Cardiomyopathy has been documented in other rodent species including woodchucks (Marmota monax), Norway rats (Rattus norvegicus) and black rats (Rattus rattus) (Roth & King, 1986). In laboratory rats, cardiomyopathy is most prevelent in older males and the severity and age of onset are influenced by a number of factors including stress, nutrition, and ambient conditions (Rothenburger et al. 2014). Obesity has also been recognised as an important risk factor for cardiomyopathy in a number of other species (Wong & Marwick, 2007). In captivity, Vancouver Island Marmots exhibit a shorter hibernation period, receive less exercise, are subject to increased hygiene and parasite control, and are fed a more protracted, uniform, and nutritionally-concentrated diet than their wild counterparts. A comparison between in situ and ex situ marmots shows that body condition indices were consistently and significantly higher in captive animals, indicating a higher level of adiposity which may be increasing their suseptibility to cardiomyopathy (Appendix H). 100 Hibernation mortalities occurred infrequently in captivity and most typically involved older marmots with identifiable health problems. Mortality records from this project support previous research indicating that death during hibernation also is an uncommon occurrence in wild, unmanipulated VIM (Bryant & Page, 2005). Low hibernation mortality also has been documented for other species, including the Olympic marmot (Marmota olympus) (Griffin et al. 2008). Differentiating between hibernation mortality and the mortality that occurs shortly before or after hibernation is difficult, and verification of high winter survival in both Vancouver Island and Olympic marmots required intensive monitoring using radiotelemetry. Conversely, hibernation mortality appeared to be significant in marmots released from captivity. Records from this project indicated that 76 of the 80 hibernation mortalities in captive-release marmots occurred during the first winter following release. Reduced overwinter survival during the first hibernation may represent a significant limiting factor for the marmot reintroduction program (Jackson, 2005). Captive VIM typically have shorter hibernations than wild marmots, and the difference in duration can be several weeks or longer (Bryant & McAdie, 2003). Although marmot life cycles are controlled by endogenous, circannual rhythms, these can be readily altered by certain exogenous factors, such as temperature, photoperiod, and food availability. Modification of endogenous cycles has the potential to disrupt growth, patterns of food consumption, reproduction and hibernation and these asynchronous cycles can be entrained for an extended period of time (Concannon et al. 1997). It is possible that captive-release marmots are released with aberrant endogenous cycles and as a result have disrupted hibernation patterns that compromise their first hibernation in the wild. For wild and captive-release VIM, predation represented the most significant cause of mortality. Predation has been implicated as an important factor in the 101 original population declines of this species (Bryant & Page, 2005; Janz et al. 2000) and as a significant cause of mortality in captive-release marmots (Aaltonen et al. 2009). During the original population declines that occurred in the late 1980s and 1990s, Wolves, cougars, and eagles were thought to represent important predators of VIM (Janz, et al. 2000). Aaltonen, et al. 2009 concluded that cougars and wolves were the most important predator for wild marmots and that Golden Eagles represented the most significant predator for captive-release marmots. In recent years, the influence of the respective predators appears to have changed. No wolf predations of telemetered marmots have been recorded since 2009 and no mortalities from Golden Eagles have been documented since 2014. During the 1990s, adult and juvenile Golden Eagles were routinely observed at active and extirpated marmot colonies, but sightings by field staff in recent years have become quite uncommon (Marmot Recovery Foundation inventory records). Over the last eight years, cougars appear to represent the biggest predation threat to be detected in wild and captive-release marmots (Figure 4). Physical examinations conducted by experienced veterinarians, necropsies performed by veterinary pathologists, and observations made by field staff offer reliable and robust health-related data. Based upon this study, the systematic collection and evaluation of these data allows for the identification and characterization of potential patterns, clusters, or syndromes and the recognition of potential health and health risk patterns in the VIM. Although the captive population was characterized by increased longevity, it also exhibited a greater number of clinical syndromes, not all of which were age-related. Intensive management and support in captivity may allow some affected animals to survive for a longer period of time than their wild counterparts, and potentially reproduce. If any of these conditions have a genetic basis, there is a potential risk that these 102 characteristics will be perpetuated within the captive population. Captive-release marmots exhibited poor survival in their first wild hibernation. More rigorous management of food availability, light and temperature regimes in captivity (that more closely mimic natural cycles) may help to normalize hibernation patterns and increase post-release survival. There was very little evidence of clinical disease in the free-ranging marmots that were examined following capture for implant surgery or translocation. However, this population had very few older individuals and opportunities to evaluate them through physical and post mortem examinations were very limited. Despite this limitation, site investigations were able to identify potential causes of mortality in free-ranging marmots that could be influencing marmot recovery. Predation continued to represent the most significant cause of mortality in freeranging marmots and recent declines in the wild population may indicate that it is still too small to overcome ongoing predation pressure. Predator-pits have been described in other species including lemmings and rabbits (Calvete et al. 1997; Krebs, 1996) and it is possible that enhanced augmentation or predator management may be necessary for the VIM to overcome the current effects of predation. With endangered species like the VIM, it is possible that health events with significant implications may only be expressed in a small number of individuals and may therefore lack numerical or statistical robustness. In addition, case documentation often involves the collection of incomplete or discrepant data. Syndromic surveillance appears to provide a conceptual framework for the ongoing characterization and comparison of these case limited or incomplete health events, and facilitates detection, evaluation and tracking of patterns or clusters as they are occurring. This allows for filtering and efficient transfer of health information so that ongoing risks can be identified and further investigated, and so that timely, 103 evidence-based decisions can be made. It is important to recognize that for this approach to be effective in the long-term, the monitoring process needs to be continual and dynamic in nature, so that new data originating from the multitude of sources are properly integrated, contextualized, and adapted with respect to previously identified events and patterns. The range of potential influences and risks elucidated in this study support the importance of continued surveillance with respect to the health management and recovery of the Vancouver Island Marmot. 104 LITERATURE CITED Aaltonen, K., Bryant, A. A., Hostetler, J. A., & Oli, M. K. (2009). Reintroducing endangered Vancouver Island Marmots: survival and cause-specific mortality rates of captive-born versus wild-born individuals. Biological Conservation 142, 2181-2190. Bryant, A. A. (1998). Metapopulation ecology of Vancouver Island Marmots (Marmota vancouverensis). Victoria: University of Victoria. Bryant, A. A., & McAdie, M. L. (2003). Hibernation ecology of wild and captive Vancouver Island Marmots (Marmota vancouverensis). Adaptive Strategies and Diversity in Marmots (pp. 149-156). Montreux: International Marmot Network. Bryant, A. A., & Page, R. E. (2005). Timing and causes of mortality in the endangered Vancouver Island Marmot (Marmota vancouverensis). Canadian Journal of Zoology (83), 674-682. Calvete, C., Villafuerte, R., Lucientes, J., & Osacar, J. J. (1997). Effectiveness of traditional wild rabbit restocking in Spain. Journal of Zoology 241 (2), 271-277. Concannon, P., Roberts, P., Baldwin, B., & Tennant, B. (1997). Long-term entrainment of circannual reproductive and metabolic cycles by northern and southern hemisphere photoperiods in Woodchucks (Marmota monax). Biology of Reproduction 57, 1008-1015. de With, N. I., Stephen, C., Ribble, R., Schwantje, H., & McAdie, M. (1999). The potential role of Yersinia spp. in the decline of the Vancouver Island Marmot (Marmota vancouverensis). Nanaimo: Centre for Coastal Health. Deem, S. L., Parker, P. G., & Miller, R. E. (2008). Building bridges: connecting the health and conservation professions. The Journal of Tropical Biology and Conservation, 662-665. Griffin, S. C., Taper, M. L., Hoffman, R., & Mills, L. S. (2008). The case of the missing marmots: are metapopulation dynamics or range-wide declines responsible? Biological Conservation 141, 1293-1309. Hart, B. L. (1988). Biological basis of the behavior of sick animals. Neuroscience & Biobehavioral Reviews 12 (2), 123-127. 105 IUCN/SSC Guidelines for Reintroductionsand Other Conservation Translocations. Version 1.0. Gland, Switzerland: IUCN Species Survival Commission IUCN Technical Guidelines on the Management of Ex-situ Populations for Conservation. (2002). Retrieved from ICUN: http://www.iucn.org/about/work/programmes/species/publications/iucn_gui delines_and__policy__statements/ Jackson, C. L. (2005). First year fidelity and survival in reintroduced captive-bred Vancouver Island Marmots (Marmota vancouverensis). Victoria: University of Victoria. Jackson, C.; Baker, A.; Doyle, D.; Franke, M.; Jackson, V.; Lloyd, N.; McAdie, M.; Stephens, T.; Traylor-Holzer, K.;. (2015). Vancouver Island Marmot Population and Habitat Viability Assessment Workshop Final Report. Apple Valley, MN: IUCN SSC Conservation Breeding Specialist Group. Janz, D. W., Bryant, A. A., Dawe, N. K., Schwantje, H., Harper, B., Nagorsen, D., . . . Simmons, R. (2000). National Recovery Plan for the Vancouver Island Marmot (Marmota vancouverensis) 2000 Update: RENEW Report No.19. Ottawa, Ontario: Environment Canada. Krebs, C. J. (1996). Population cycles revisited. Journal of Mammalogy 77(1), 8-24. Matthews, F., Orros, M., McLaren, G., Gelling, M., & Foster, R. (2005). Keeping fit on the ark: assessing the suitability of captive-bred animals for release. Biological Conservation 121, 569-577. Naughton, D. (2012). The Natural History of Canadian Mammals. Canadian Museum of Nature and University of Toronto Press, Toronto, Ontario. Ranheim, B., Rosell, F., Haga, H. A., & Arnemo, J. M. (2004). Field anaesthetic and surgical techniques for implantation of intraperitoneal radio transmitters in Eurasian beavers (Castor fiber). Wildlife Biology 10 (1), 11-15. Raverty, S., & Black, S. (2001). Causes of death in captive Vanvouver Island Marmots (Marmota vancouverensis) including presumptive mycoplasmosis. Canadian Veterinary Journal 42, 386-387. 106 Roth, L., & King, J. M. (1986). Congestive cardiomyopathy in the Woodchuck (Marmota monax). Journal of Wildlife Disease 22 (4), 553-537. Rothenburger, J. L., Himsworth, C. G., Treuting, P. M., & Leighton, F. A. (2014). Survey of cardiovascular pathology in wild urban Rattus norvegicus and Rattus rattus. Veterinary Pathology Online. Soto-Azat, C., Boher, F., Fabry, M., Medina-Vogel, G., & Pascual, P. (2008). Surgical implantation of intra-abdominal radio-transmitters in Marine Otters (Lontra felina) in Central Chile. Journal of Wildlife Diseases 44 (4), 979-982. Swarth, H. S. (1911). Two new species of marmots from North America. University of California Publications in Zoology (7), 201-204. Swarth, H. S. (1912). Report on a collection of birds and mammals from Vancouver Island. University of California Publications in Zoology (10), 1-124. Van Vuren, D. (1989). Effects of intraperitoneal implants on yellow-bellied marmots. Journal of Wildlife Management. 53 (2), 320-323. Watson, J. (1997). The Golden Eagle. London: T & A D Poyser. Wild, P. (2013). The Cougar: Beautiful, Wild and Dangerous. Madeira Park: Douglas and McIntyre. Wong, C., & Marwick, T. H. (2007). Obesity cardiomyopathy: pathogenesis and pathophysiology. Nature Reviews Cardiology 4, 436-443. 107 CHAPTER 4 CONCLUSION The overarching goal of this thesis was to expand upon our understanding of individual and population health in the critically endangered Vancouver Island Marmot (Marmota vancouverensis). My approach was to describe and evaluate select clinical and pathological data originating from a variety of sources. This involved the analysis of 1,106 Vancouver Island Marmot (VIM) blood profiles, 3,174 physical examinations, 140 necropsies, and 533 field mortality records. In Chapter 2, I calculated hematology and serum biochemistry reference ranges as a baseline metric for assessing VIM health. I also determined that VIM blood values are qualitatively comparable to those that have been calculated for other rodent species and that certain leukogram and plasma protein values may have potential utility as a quantitative measure for comparing VIM management groups. In Chapter 3, I use physical examinations, post mortem examinations, and field evaluation of marmot mortalities, to identify health trends in captive, captive-release and wild VIM. These analyses sought to determine what is expected for this species under different conditions (wild, captive and captive-release) and to identify potential risk factors that may be influencing the species’ capacity to achieve or maintain health and to meet defined recovery objectives. DEFINING HEALTH IN THE VANCOUVER ISLAND MARMOT The relative state of health of a wildlife population is not necessarily defined by a discrete set of biological characteristics but by human expectations and constructs (Stephen, 2014) which seek to identify an ideal standard of population well-being (Hanisch, et al. 2012). With the formulation of officially recognized 108 conservation strategies, the VIM’s health status is determined by the species’ relative capacity to fulfill discrete recovery objectives with specific numerical, spatial, and temporal components. Evaluating the population’s health includes the identification and assessment of determinants and outcomes which are compatible with, or which compromise, the species’ capacity for achieving normative recovery goals. VIM health is contingent upon many factors including the attributes of the animals, their environment, the presence or absence of tangible, effective recovery actions, and the logistical and socio-economic feasibility of implementing these actions. THERE IS NOT ONE HEALTH FOR VIM It is important to recognize that health in the Vancouver Island Marmot cannot be characterized by a singular, uniform state. Recovery efforts for VIM have involved an intensive program of captive breeding and conservation translocations, requiring the monitoring and management of several groups or subpopulations. These include; (A) the original in situ population that was under threat and demonstrating dramatic declines, (B) the captive founder population consisting of wild-born individuals captured and transitioned to the artificial conditions of captivity, (C) the ex situ population comprised of captive-born animals raised under the artificial conditions of captivity, (D) the captive-release population consisting of captive individuals transitioning back to the wild, and (E) the free-ranging recovery population consisting of successful captive-release marmots, their progeny and the descendents of the original wild marmots. Each of these groups has been subject to a unique set of natural and artificial determinants which have influenced health outcomes and expectations. There are also significant differences in the extent to which each of these groups can be monitored and manipulated. However, from a conservation and management perspective, the relative state of health of the 109 subpopulations will collectively influence the species’ capacity to achieve recovery objectives. If the health of one group is compromised, it has the potential to jeopardise the integrity of the other groups and affect species recovery overall. The original in situ population In the 1980s and 1990s, there was dramatic destabilization of the VIM population and it suffered severe declines. As a result, the wild population was no longer autonomous and self sustaining, and it was deemed to be incapable of independently achieving recovery targets (Janz, et al., 2000). The original population declines had been attributed to demographic and ecosystem effects (Janz, et al., 2000), but there was insufficient historical data for this analysis to consider whether changes to marmot health may also have been a contributing factor. The ex situ population Captivity is not entirely benign and artificial conditions posed a novel set of risk factors for VIM, particularly as management and husbandry practices were in their initial stages of development. Regular observation, routine clinical evaluation, and thorough post mortem examination of captive individuals resulted in the description of a number of health conditions not identified in any free-ranging marmots. For example, intensive management and protection in captivity appears to have led to increased marmot longevity, allowing for the appearance of age-related problems such as acquired cardiovascular disease and neoplasia, which collectively accounted for approximately 50% of captive mortalities. As captive marmots age, it is possible that obesity may represent an increased risk factor for these disorders. Although there were congenital defects identified in the captive population, they did not occur with enough frequency or predictability to suggest a progressive increase in the number of genetically compromised individuals or in the level of inbreeding (Pimm et al., 2006; Raikkonen et al., 2006). None of the individuals 110 displaying congenital disorders were allowed to breed in captivity and since 2011 no congenital problems have been been identified in the captive population. It is possible that captive support may have prolonged the survival of young marmots suffering from these abnormalities. Overall, the captive population has proven effective in meeting its defined objectives, which includes acting as a long-term safeguard, a genetic reservoir, and a provider of marmots for release. The captive program’s future health, and its capacity for continuing to achieve recovery targets, appears to be dependent upon the continued maintenance of effective biosecurity measures and a suitable captive population size, which allows for the proper maintenance of genetic integrity. A viable captive program is dependent upon political will and adequate resources. The captive-release and free-ranging recovery population Captive-release marmots have been documented to have lower survival than their wild-born counterparts, and this is most apparent in their first year following release from captivity (Aaltonen et al., 2009). The first hibernation in the wild represented a significant cause of mortality and it is possible that captive marmots are released with shortened or abberent endogenous cycles which may compromise their initial hibernation. There are limited data on the fall body mass of captiverelease marmots, but it is also possible that poor body condition at the time of immergence or emergence could also jeopardise overwinter survival. Captiverelease marmots that survive their first winter in the wild exhibit subsequent hibernation survival that is comparable to their wild counterparts. Although marmots released from captivity do survive and facilitate growth of the free-ranging population (Jackson et al., 2015), it is possible that this contribution could be significantly enhanced by increasing survival during their first hibernation. 111 Although captive-release marmots were not routinely trapped and examined during their first year following release, they were opportunistically captured, examined, and sampled in subsequent years. In terms of physical examinations, body condition, bloodwork and occurance of morbidity, these surviving captiverelease marmots were clinically comparable to their wild-born counterparts. Overall, there was a paucity of health conditions identified in free-ranging marmots (both wild and captive-release) compared to their captive counterparts. This could be due to a fundamental lack of disease, or limited opportunities to observe or examine compromised marmots due to their reclusive behaviors or poor survival. In addition to not being observed or evaluated with the same intensity, in situ marmots did not appear to exhibit the same longevity as ex situ animals, thereby limiting the appearance of age-related disorders. For wild and captive-release Vancouver Island marmots, predation represented the most common cause of mortality. In each of these two groups, predation accounted for at least 95% of the non-hibernation deaths for which a specific cause was identified. Predation has been implicated as an important factor in the original population declines of the species (Bryant & Page, 2005; Janz et al. 2000) and as a significant cause of mortality in captive-release marmots (Aaltonen et al. 2009). Mortality records current to the end of the 2017 field season indicate that predation continues to be a limiting factor in marmot population growth and recovery. Even though the wild population has shown encouraging growth associated with reintroductions and translocations, it may still be too small to overcome the inhibiting effects of ongoing predation pressure. Similar situations have been described for a range of species including lemmings and rabbits (Calvete et al. 1997; Krebs, 1996). Like the original in situ population, the current recovery 112 population lacks autonomous sustainability and remains dependent upon captive augmentation to achieve defined recovery goals. INFECTIOUS DISEASE Infectious agents have been identified as an important cause of declines in many endangered populations (Biedrzycka & Kloch, 2016) and have been suggested as a potential threat to VIM (Jackson et al. 2015; Janz et al. 2000; Bryant, 1998). There are many examples of rare species being affected by infectious disease, including chytridiomycosis in amphibians, canine distemper in Serengeti lions (Panthera leo), Ethiopian wolves (Canis simensis) and black-footed ferrets (Mustela nigripes), feline leukemia virus in Iberian Lynx (Lynx pardinus) and Plasmodium sp. in Hawaiin land birds (Gordon, et al., 2015; Roznik & Alford, 2015; Ewan et al., 2012; Cleaveland, 2009; Thorne & Williams, 1998). With respect to VIM, this analysis of clinical and pathological data did not identify any specific infectious agents in captive or freeranging marmots that represented a generalized population threat. However, there is always the ongoing risk of future exposure to a novel pathogen. Zoological facilities maintain eclectic collections of multiple taxa from a diversity of sources, each with their own spectrum of infectious agents (Snyder, et al., 1996). Captive facilities may also inadvertently harbour pest species which provide an additional reservoir for introduction of exotic pathogens. Although captive VIM are maintained under strict biosecurity protocols, quarantine procedures may be compromised whenever marmots are transferred between facilities or translocated. Also, infectious agents may emerge or be maintained in sympatric or introduced species on Vancouver Island. The threat of novel infectious agents cannot be quantified because it is impossible to delineate the full spectrum of VIM’s suseptibility to infectious agents. This susceptibilty could potentially increase if 113 existing biosecurity measures are relaxed, or if the population experiences additional loss of genetic variation and immunocompetence due to a decrease in population size (Biedrzycka & Kloch, 2016). RECOMMENDATIONS • Although most of the current threats to the VIM population appear to be associated with anthropogenic or ecological factors, artificial management, low genetic diversity, and small numbers place the species at additional risk for introduction of novel infectious agents and genetic deterioration. Although this risk cannot be quantified, continued health surveillance of the captive and freeranging populations, using the data from this thesis as a baseline for comparison, will help to recognize future changes and identify potential threats. • It is possible that existing biosecurity measures in captivity may have acted as an effective safeguard against the introduction of novel infectious diseases. These precautions are well integrated into existing management protocols and should be continued. • Obesity may increase the risk for age-related disorders in captive marmots, including cardiovascular disease and neoplasia. Management practices, including diet composition and seasonal duration of feeding, and how they influence body condition and hibernation, should be reviewed. • It is possible that congenital problems and mange may be indicative of genetic compromise or inbreeding in VIM. Although the occurrence of these disorders appears to have abated over time, marmots should be carefully monitored for their reappearance. In general, marmots with congenital problems should not be allowed to breed and marmots exhibiting mange should not be allowed to breed to closely related individuals. 114 • The first wild hibernation results in significant mortality of captive-release marmots. Although marmots are confirmed to have good body reserves at the time of their release from captivity, it is possible that they may lose condition as they transition to a wild environment. This loss may compromise their ability to survive their first hibernation. There is a need for further research which compares the hibernation characteristics of captive, captive-release, and wild marmots and investigates the effects of entrainment of endogenous cycles in captivity, marmot body condition at immergence and emergence, hibernacula characteristics and the benefits of pre and post-hibernation supplementation. • From 1992 to 2016 a total of 898 implant surgeries were performed on VIM including 96 replacement surgeries. Two fatal complications (0.22% of total surgeries) have arisen directly from implanted transmitters. In one case, the radio-transmitter become lodged in the pelvic canal of an emaciated postemergent male, resulting in a fatal impaction of the gastrointestinal tract. In the second case, the outer surface of the transmitter caused an extreme, chronic inflammatory reaction with significant encapsulation and visceral adhesions. These problems were subsequently mitigated by having the manufacturer increase the diameter of the transmitters and by having the transmitters encapsulated in a hard, biologically inert resin. Although there were focal adhesions to the greater omentum associated with the early use of wax-coated transmitters (1992 to 2010, Appendix D), there was no corrosion, leakage or overt breakdown of the radio-transmitters as has been reported in other species (Arnemo et al., 2018). Based upon bloodwork, physical examinations (including post-surgical evaluations) and necropsies, the implantation of abdominal radiotransmitters does not appear to impact marmot health, and these units should continue to be used to monitor free-ranging marmots. They represent the most 115 effective method for monitoring survival and mortalities in the free-ranging population. • Although mortalities in free-ranging marmots are generally characterized by limited remains and forensic clues, these events should continue to be rigorously investigated as part of an expanding data set. • Although recovery of intact marmot carcasses from the field is a rare occurrence, these events represent significant clinical data and should be comprehensively evaluated as opportunities present themselves. THESIS SIGNIFICANCE My thesis extended our understanding of health in the Vancouver Island Marmot by collating select clinical and pathological data from multiple sources. It described characteristics for blood values, morbidity, and mortality under different management treatments (wild, captive and captive-release) and identified potential threats to both captive and free-ranging marmots. Although these results are important, they are by no means definitive in nature. This analysis is not meant to represent an exhaustive or conclusive treatment of VIM health data. The greatest utility of this thesis is that it can serve as a template for ongoing comparisons and for recognizing change. Marmot health and its influences are complex and dynamic in nature, and therefore continued health surveillance and evaluation of existing and new data represent important keys for effective management and species recovery. 116 LITERATURE CITED Arnemo, J., Ytrehus, B., Madslien, K., Maimsten, J., Brunberg, S., Segerstrom, P., Evans, A. L., & Swenson, J. E. (2018). Long-term safety of intraperitoneal radio transmitter implants in brown bears (Ursus arctos). Frontiers in Veterinary Science 5: 252. Aaltonen, K., Bryant, A. A., Hostetler, J. A., & Oli, M. K. (2009). Reintroducing endangered Vancouver Island marmots: survival and cause-specific mortality rates of captive-born versus wild-born individuals. Biological Conservation 142, 2181-2190. Biedrzycka, A., & Kloch, A. (2016). Development of novel associations between MHC alleles and susceptibility to parasitic infections in an isolated population of an endangered mammal. Infection, Genetics and Evolution 44, 210-217. Bryant, A. A., & Page, R. E. (2005). Timing and causes of mortality in the endangered Vancouver Island marmot (Marmota vancouverensis). Canadian Journal of Zoology (83), 674-682. Bryant, A. A. (1998). Metapopulation ecology of Vancouver Island marmots (Marmota vancouverensis), PhD thesis. Victoria, B.C.: University of Victoria. Calvete, C., Villafuerte, R., Lucientes, J., & Osacar, J. J. (1997). Effectiveness of traditional wild rabbit restocking in Spain. Journal of Zoology 241 (2), 271-277. Cleaveland, S. (2009). Viral threats and vaccination: disease management of endangered species. Animal Conservation 12, 187-189. Ewan, J. C., Armstrong, D. P., Parker, P. A., & Seddon, P. J. (2012). Reintroduction Biology: Integrating Science and Management. Wiley- Blackwell. Gordon, C. H., Banyard, A. C., Hussein, A., Laurenson, M. K., Malcolm, J. R., Marino, J., . . . Sillero-Zubiri, C. (2015). Canine distemper in endangered Ethiopian wolves. Emerging Infectious Diseases 21 (5), 824-832. 117 Jackson, C.; Baker, A.; Doyle, D.; Franke, M.; Jackson, V.; Lloyd, N.; McAdie, M.; Stephens, T.; Traylor-Holzer, K.;. (2015). Vancouver Island Marmot Population and Habitat Viability Assessment Workshop Final Report. Apple Valley, MN: IUCN SSC Conservation Breeding Specialist Group. Janz, D. W., Bryant, A. A., Dawe, N. K., Schwantje, H., Harper, B., Nagorsen, D., . . . Simmons, R. (2000). National Recovery Plan for the Vancouver Island Marmot (Marmota vancouverensis) 2000 Update: RENEW Report No.19. Ottawa, Ontario: Environment Canada. Krebs, C. J. (1996). Population cycles revisited. Journal of Mammalogy 77(1), 8-24. Pimm, S. L., Dollar, L., & Bass Jr., O. L. (2006). The genetic rescue of the Florida pather. Animal Conservation 9, 115-122. Raikkonen, J., Bignert, A., Mortensen, P., & Fernholm, B. (2006). Congenital defects in a highly inbred wild wolf population (Canis lupus). Mammalian Biology 71 (2), 65-73. Roznik, E. A., & Alford, R. A. (2015). Seasonal ecology and behavior of an endangered rainforest frog (Litoria rheocola) threatened by disease. PloS One 10(5): e127851.doi:10.137/journal, 1-17. Snyder, N. F., Derrickson, S. R., Bessinger, S. R., Wiley, J. W., Smith, T. B., Toone, W. D., & Miller, B. (1996). Limitations of captive breeding in endangered species recovery. Conservation Biology 10 (2), 338-348. Stephen, C. (2014). Toward a modernized defintion of wildlife health. Journal of Wildlife Diseases 5 (3), 427-430. Thorne, E. T., & Williams, E. S. (1998). Disease and endangered species: the blackfooted ferret as a recent example. Conservation Biology 2(1), 66-74. 118 Wild pup captures (45) Total wild captures (71) Weaned captive litters (180) Weaned captive pups (612) Weaned males (56%) Weaned females (44%) Unknown Average weaned litter size Captive Mortalities (113) Releases of captive marmots (515) Recaptures (2) Captive Total (58) 4 2 6 0 0 0 0 0 0.00 0 0 0 6 1998 5 3 8 0 0 0 0 0 0.00 2 0 0 12 1999 9 10 19 0 0 0 0 0 0.00 4 0 0 27 2000 1 4 5 2 8 5 3 0 4.00 1 0 0 39 2001 2 5 7 1 2 1 1 0 2.00 1 0 0 47 2002 2 4 6 5 13 8 5 0 2.60 3 0 0 63 2003 1 2 3 7 18 10 8 0 2.57 4 4 1 77 2004 0 1 1 8 26 19 7 0 3.25 2 9 0 93 2005 0 0 0 13 26 0.50 48 31 17 0 3.69 5 15 0 121 2006 0 0 0 14 30 0.47 55 27 28 0 4.00 4 31 1 142 2007 0 0 0 15 37 0.41 60 33 27 0 4.00 3 37 0 162 2008 0 0 0 23 46 0.50 85 41 40 4 3.70 11 59 0 177 2009 0 0 0 20 48 0.42 71 33 38 0 3.55 9 68 0 171 2010 0 0 0 18 49 0.37 55 33 22 0 3.06 11 85 0 130 2011 0 0 0 18 36 0.50 51 26 24 1 2.83 8 66 0 107 2012 0 0 0 6 26 0.23 22 11 11 0 3.66 9 34 0 86 2013 0 0 0 6 25 0.24 18 11 7 0 3.00 6 16 0 82 2014 0 0 0 6 19 0.36 18 10 8 0 2.83 16 29 0 55 2015 0 0 0 5 12 0.42 15 9 6 0 3.00 1 24 0 45 2016 1 5 6 3 14 0.21 13 8 5 0 4.33 9 13 0 42 2017 0 2 2 5 11 0.45 19 12 7 0 3.80 3 11 0 49 2018 1 8 9 5 12 0.42 15 11 4 0 3.00 1 14 0 58 Total 26 46 72 180 391 0.40 612 339 268 5 3.40 113 515 2 58 Proportion of Pairs Breeding (2005 to 18) Wild marmot captures excluding pups (26) 1997 Breeding Pairs (from 2005 to 2018) Year Appendix A. Vancouver Island Marmot (Marmota vancouverensis) captive population numbers from 1997 to 2018. 118 119 Appendix B. Descriptive statistics for hematological variables from clinically normal or healthy Vancouver Island Marmots (Marmota vancouverensis) before & after removal of outliers. Used in calculation of reference values in accordance with guidelines established by Species 360, formerly the International Species Information System (Teare, Mean Min Max N S.D. 3 x S.D. minus 3 x S.D. plus 3 x S.D. Number of outliers Recalculated Mean Min Max N White blood cells WBC x 10⁹/L 5.1 0.6 35.9 1030 2.4 7.1 -2.0 12.2 12.0 5.0 0.6 12.2 1018 1.9 Segmented neutrophils Neutro x 10⁹/L 2.7 0.3 26.6 1027 1.6 4.8 -2.1 7.6 10.0 2.6 0.3 7.6 1017 1.2 Band neutrophils Bands x 10⁹/L 0.0 0.0 0.5 1027 0.0 0.1 -0.1 0.1 15.0 0.0 0.0 0.0 1012 0.0 Lymphocytes Lympho x 10⁹/L 2.0 0.0 12.0 1027 1.2 3.7 -1.7 5.6 9.0 1.9 0.0 5.5 1018 1.1 Monocytes Mono x 10⁹/L 0.3 0.0 6.1 1027 0.4 1.1 -0.7 1.4 15.0 0.3 0.0 1.4 1012 0.3 Eosinophils Eosino x 10⁹/L 0.0 0.0 1.1 1027 0.1 0.2 -0.2 0.3 16.0 0.0 0.0 0.2 1011 0.0 Basophils Baso x 10⁹/L 0.0 0.0 0.3 1027 0.0 0.1 -0.1 0.1 24.0 0.0 0.0 0.1 1003 0.0 Parameter S.D. Units 2002). WHITE BLOOD CELLS 119 120 Appendix B (cont.) Descriptive statistics for hematological variables from clinically normal or healthy Vancouver Island Marmots (Marmota vancouverensis) before & after removal of outliers. Used in calculation of reference values in accordance with guidelines established by Species 360, formerly the International Species Information System. Mean Min Max N S.D. 3 x S.D. minus 3 x S.D. plus 3 x S.D. Number of outliers Recalculated Mean Min Max N Red blood cells RBC x 10 ¹²/L 6.4 2.8 8.9 1025 0.7 2.2 4.3 8.6 15.0 6.4 4.2 8.5 1010 0.6 Hemoglobin Hb g/L 144.4 15.0 198.0 1016 16.2 48.7 95.7 193.0 12.0 145.1 98.0 187.0 1004 14.3 Hematocrit Hmt L/L 0.4 0.2 0.6 1030 0.0 0.1 0.3 0.6 9.0 0.4 0.3 0.6 1021 0.0 MCV fl 65.8 5.1 92.7 992 5.6 16.7 49.1 82.5 15.0 65.9 50.0 82.3 977 3.5 MCH pg 22.4 16.3 33.1 986 1.6 4.8 17.6 27.2 0.0 22.4 16.3 33.1 986 1.6 MCHC g/L 339.5 253.0 493.0 996 15.3 46.0 293.6 385.5 13.0 339.3 294.0 384.0 983 12.3 RCDW %CV 14.3 5.2 30.3 904 2.1 6.4 7.9 20.7 27.0 14.4 10.1 20.3 877 1.5 Platelet count x 10⁹/L 323.0 37.0 1295.0 767 104.9 314.6 8.3 637.6 6.0 318.7 37.0 625.0 761 91.7 Mean platelet volume fl 8.9 0.0 16.9 704 1.4 4.3 4.6 13.2 10.0 8.9 6.7 14.0 694 1.3 Parameter S.D. Units (Teare, 2002) ERYTHROCYTES Mean corpuscular volume Mean corpuscular hemoglobin Mean corpuscular hemoglobin concentration Red cell distribution width HEMOSTASIS 120 121 Appendix B (cont.) Descriptive statistics for hematological variables from clinically normal or healthy Vancouver Island Marmots (Marmota vancouverensis) before & after removal of outliers. Used in calculation of reference values in accordance with guidelines established by Species 360, formerly the International Species Information System. Mean Min Max N S.D. 3 x S.D. minus 3 x S.D. plus 3 x S.D. Number of outliers Recalculated Mean Min Max N Sodium mmol/L 143.7 120.0 164.0 532 4.6 13.9 129.8 157.7 7.0 143.8 130.0 155.8 525 4.0 Potassium mmol/L 5.6 3.3 50.0 531 2.8 8.5 -2.9 14.1 9.0 5.4 3.3 13.3 522 1.4 Sodium / Potassium ratio ratio 27.9 3.0 43.6 531 6.5 19.4 8.5 47.3 6.0 28.1 8.6 43.6 525 6.1 Chloride mmol/L 102.0 18.0 112.0 529 5.5 16.5 85.5 118.5 1.0 102.1 88.0 112.0 528 4.1 Bicarbonate mmol/L 26.4 9.0 39.0 147 5.9 17.6 8.7 44.0 0.0 26.4 9.0 39.0 147 5.9 Carbon Dioxide mmol/L 26.4 5.0 38.0 357 5.6 16.8 9.6 43.2 0.0 26.4 5.0 38.0 357 5.6 20.9 6.0 75.9 515 7.1 21.4 -0.5 42.3 3.0 20.7 6.0 41.0 512 6.6 Parameter S.D. Units (Teare, 2002) ELECTROLYTES AND ACID-BASE Anion Gap Calcium mmol/L 2.4 1.4 22.0 601 0.9 2.7 -0.2 5.1 3.0 2.4 1.4 3.3 598 0.2 Phosphorous mmol/L 1.9 0.1 5.8 602 0.5 1.6 0.2 3.5 8.0 1.8 0.7 3.5 594 0.5 Calcium / Phosphorous ratio ratio 1.5 0.4 28.3 576 1.3 3.9 -2.5 5.4 3.0 1.4 0.4 4.3 573 0.4 Calculated Osmolality mmol/kg 297.5 265.0 355.0 514 7.7 23.1 274.5 320.6 6.0 297.3 276.0 319.0 508 6.7 121 122 121 Appendix C. Descriptive statistics for serological variables from clinically normal or healthy Vancouver Island Marmots (Marmota vancouverensis) before & after removal of outliers. Used in calculation of reference values in accordance with guidelines established by Species 360, formerly the International Species Information System. (Teare, Mean Min Max N S.D. 3 x S.D. minus 3 x S.D. plus 3 x S.D. Number of outliers Recalculated Mean Min Max N Total Protein g/L 62.2 6.0 107.0 652 10.2 30.7 31.6 92.9 9.0 62.0 42.0 90.0 643 9.0 Albumin g/L 27.8 17.0 84.0 566 5.2 15.6 12.2 43.3 11.0 27.3 17.0 43.0 555 3.6 g/L 33.3 18.6 75.0 561 6.9 20.8 12.4 54.1 8.0 32.9 18.6 53.0 553 6.0 ratio 0.9 0.3 10.0 563 0.5 1.5 -0.6 2.4 2.0 0.9 0.3 1.9 561 0.2 μmol/L 2.8 0.0 15.0 435 1.7 5.1 -2.3 7.9 7.0 2.6 0.0 7.0 428 1.4 Parameter S.D. Units 2002) PROTEINS Globulin Albumin / Globulin ratio A/G ratio LIVER AND MUSCLE Total Bilirubin ALP IU/L 77.2 5.0 1101.0 564 68.7 206.2 -129.1 283.4 8.0 71.6 5.0 266.0 556 41.3 Alanine Aminotransferase ALT IU/L 23.1 1.0 314.0 526 33.4 100.3 -77.1 123.4 14.0 18.6 1.0 117.0 512 17.0 Aspartate Aminotransferase AST IU/L 43.9 1.0 601.0 458 61.3 183.9 -139.9 227.8 9.0 37.1 1.0 222.0 449 34.5 Gamma Glutamyltransferase GGT IU/L 4.5 0.0 55.0 526 5.1 15.3 -10.8 19.8 10.0 4.0 0.0 18.0 516 3.0 Creatinine Phosphokinase CPK IU/L 640.5 56.0 10748.0 595 924.8 2774.5 2134.0 3414.9 12.0 526.2 56.0 2970.0 583 366.2 mmol/L 7.2 3.7 13.1 72 2.0 5.9 1.3 13.2 0.0 7.2 3.7 13.1 72 2.0 mmol/L 8.8 1.2 18.3 637 2.4 7.3 1.5 16.1 7.0 8.8 2.2 15.8 630 2.3 Cholesterol Glucose Gluc 122 Alkaline Phosphatase 123 121 Appendix C (cont.) Descriptive statistics for serological variables from clinically normal or healthy Vancouver Island Marmots (Marmota vancouverensis) before & after removal of outliers. Used in calculation of reference values in accordance with guidelines established by Species 360, formerly the International Species Information System. (Teare, Mean Min Max N S.D. 3 x S.D. minus 3 x S.D. plus 3 x S.D. Number of outliers Recalculated Mean Min Max N Blood Urea Nitrogen BUN mmol/L 11.1 3.1 70.0 638 3.8 11.4 -0.3 22.6 6.0 10.9 3.1 22.2 632 2.6 Creatinine Creat μmol/L 82.5 7.0 224.0 623 23.6 70.7 11.8 153.3 6.0 81.9 18.0 150.0 617 21.8 ratio 0.1 0.0 1.0 623 0.1 0.2 -0.1 0.4 10.0 0.1 0.0 0.4 613 0.1 IU/L 1785.0 865.0 4068.0 30 822.3 2466.9 681.9 4251.8 0.0 1785.0 865.0 4068.0 30 822.3 Amylase IU/L 878.3 147.0 2373.0 79 481.8 1445.3 567.0 2323.5 2.0 839.8 147.0 2086.0 77 423.0 Lipase IU/L 191.8 60.0 856.0 73 137.2 411.7 219.9 603.5 0.0 191.8 60.0 856.0 73 137.2 Tetra Iodothyronine nmol/L 57.5 20.0 112.0 152 17.3 52.0 5.5 109.6 1.0 57.2 20.0 91.0 151 16.8 Parameter S.D. Units 2002) RENAL FUNCTION Bun / Creatinine ratio OTHER Lactose Dehydrogenase LDH 123 124 Appendix D. Characteristics of very high frequency (VHF) implantable radiotransmitter used in Vancouver Island Marmots (Marmota vancouverensis) from 50 1095 1095 Non-pups Non-pups Years Age Category 40 Temperature Responsive Pulse Rate 100 Maximum Longevity (days) Length (mm) Diameter (mm) 23 85 35 Duty Cycle IMP/325/L 23 90 Pups and non-pups Preparation Telonics® IMP/300/L 25 Not recorded Encapsulation Material Telonics® Not recorded Weight (grams) Custom Telemetry® Model Manufacturer 1992 to 2018. Wax Disinfected povidoneiodine solution None Continuous temperature response 1992 1993 Wax Disinfected povidoneiodine solution 9 hours on 15 hours off Wax Disinfected povidoneiodine solution 9 hours on 15 hours off 9 hours on 15 hours off Temperature triggered response (35 ppm above 30°C / 25 ppm below 30°C) Temperature triggered response (35 ppm above 30°C / 25 ppm below 30°C) Temperature triggered response (35 ppm above 30°C / 25 ppm below 30°C) 1994 2009 2010 Telonics® IMP/200/L 23 61 25 540 Pups Wax Disinfected povidoneiodine solution Advanced Telemetry Systems® M1215T 12 64 13 294 Pups Resin Sterilized ethylene oxide gas None Continuous temperature response 2011 2013 Advanced Telemetry Systems® M1230T 18 68 25 728 Pups Resin Sterilized ethylene oxide gas None Continuous temperature response 2011 2013 Advanced Telemetry Systems® M1240T 20 78 40 1226 Non-pups Resin Sterilized ethylene oxide gas None Continuous temperature response 2011 2013 Holohil Systems® A1-2TH 19 79 35 1095 Non-pups Disinfected povidoneiodine solution None Continuous temperature response 2006 2009 Holohil Systems® A1-2TH 25 90 55 1800 Non-pups Sterilized ethylene oxide gas None Continuous temperature response 2009 2011 Holohil Systems® A1-2TH 25 90 50 1800 Non-pups Sterilized ethylene oxide gas None Continuous temperature response 2012 2018 Plasti Dip® (butyl rubber) Plasti Dip® (butyl rubber) Resin 2008 2010 125 Appendix D. (cont.) Characteristics of very high frequency (VHF) implantable radio-transmitter used in Vancouver Island Marmots (Marmota vancouverensis) 540 Holohil Systems® S1-2TH 15 65 18 540 Years 18 Temperature Responsive Pulse Rate 65 Duty Cycle 15 Preparation S1-2TH Encapsulation Material Holohil Systems® Age Category Maximum Longevity (days) Weight (grams) Length (mm) Diameter (mm) Model Manufacturer from 1992 to 2018. Pups Plasti Dip® (butyl rubber) Sterilized ethylene oxide gas None Continuous temperature response 2007 2011 Pups Resin Sterilized ethylene oxide gas None Continuous temperature response 2012 2013 126 Appendix E. Summary of Vancouver Island Marmot (Marmota vancouverensis) implant surgeries, 1992 to 2018. Wild marmots Captive-release marmots Year new surgeries replacement surgeries total (wild & translocation) wild population estimate % of total wild population new surgeries (prerelease) replacement surgeries (post release) Annual Total 1992 7 0 7 211 3.3 0 0 7 1993 11 2 13 189 6.9 0 0 13 1994 6 3 9 233 3.9 0 0 9 1995 4 0 4 139 2.9 0 0 4 1996 4 0 4 122 3.3 0 0 4 1997 2 0 2 118 1.7 0 0 2 1998 4 0 4 74 5.4 0 0 4 1999 3 0 3 60 5.0 0 0 3 2000 7 0 7 51 13.7 0 0 7 2001 12 2 14 43 32.6 0 0 14 2002 7 1 8 41 19.5 1 0 9 2003 3 6 9 31 29.0 3 0 13 2004 1 6 7 32 21.9 9 0 17 2005 8 3 11 40 27.5 15 0 26 2006 3 0 3 60 5.0 30 2 35 2007 9 0 9 68 13.2 37 1 47 2008 3 4 7 100 7.0 59 2 68 2009 18 3 21 160 13.1 68 4 93 2010 30 3 33 280 11.8 85 5 123 2011 12 1 13 350 3.7 67 6 86 2012 19 11 30 375 8.0 32 11 73 2013 57 8 65 313 20.8 16 0 81 2014 35 2 37 245 15.1 27 0 64 2015 26 3 29 240 12.1 24 1 54 2016 23 7 30 166 18.1 13 1 44 2017 13 4 17 150 11.3 11 33 28 2018 24 5 29 145 20.0 14 0 43 425 149 (average) 12.4 (average) 511 33 969 Total 353 72 127 Appendix F. Trapping, handling, anesthesia & implant surgery techniques in Vancouver Island Marmots (Marmota vancouverensis) Trapping Free-ranging marmots were captured using live-traps (Havahart® model # 1079, dimensions 32” x 10” x 12”) which were baited with a trail of peanut butter or set up so that they represented the marmot’s only option for egress from a burrow. Traps set for marmots were continuously monitored or checked frequently. Because of their susceptibility to hyperthermia (Ken Langelier, personal communication, 1992), marmots were not typically handled or immobilized if ambient temperatures exceeded 20C. Following capture, marmots held in live traps were covered with a fitted trap-cover to provide shade, minimize stress, and to act as a visual barrier to reduce self-trauma associated with hitting the sides of the wire trap. Marmots undergoing implantation were transported to a central field site for surgery. The distance to this site was minimized so that transport was as quick and efficient as logistically possible. Hunter pack-frames were often used to carry the trapped marmot to the handling location. In hot weather, a sealed plastic bag containing flattened snow (if available) or an ice-pack were placed beneath a portion of the trap on the pack-frame’s shelf to facilitate cooling during transport. Marmots trapped at Mount Washington were often transported by foot or by vehicle to the Tony Barrett Mount Washing Marmot Recovery Centre (MRC) for implant surgery or for examination and staging prior to translocation. Wild marmots captured for the captive breeding program were trapped, examined, and then directly transported to the Toronto Zoo (total = 26), Calgary Zoo (25) by vehicle and commercial airline, or to the MRC (10) by vehicle. 128 Handling For most procedures involving sampling, marking or examination of captive or free-ranging marmots, chemical restraint was required. Excessive physical restraint of the marmots was avoided because of the potential for traumatic injuries including diaphragmatic hernias which have occurred in woodchucks at Cornell University following physical restraint, (Tennant, personal communication, 2001) and hyperthermia. The marmots were run into a tapered, cloth handling bag which fit snugly around the end of the trap or against the doorway of their nest-box. Any obstacle such as rocks or branches lying in the “run-way” area of the cloth bag was removed beforehand to avoid injury to the marmot. In captivity, the concrete floor was padded by pulling additional shavings into the “run-way” area. Once the marmot had reached the apex of the bag, the bag was gripped or twisted behind the animal to provide better restraint and prevent its turning or egress, taking care to not grab its tail. Immobilization / Anesthesia Once they were manually restrained in the handling bag the marmots received an intramuscular injection of ketamine hydrochloride (10 mg/kg) and midazolam hydrochloride (0.25 mg/kg). Ketamine hydrochloride (100 mg/ml) was combined with midazolam hydrochloride (5 mg/ml) in a 2:1 ratio and administered at an approximate volume of 0.15 ml / kg into the epaxial or lumbar muscles, which were palpated and isolated through the wall of the bag. Following injection, care was taken so that the marmot was not excessively wrapped or physically restrained within the bag, which could increase its struggling, elevate its body temperature, or interfere with its breathing. Marmots were monitored for a suitable drug response through the wall of the bag. This included reduced struggling, progressive muscle relaxation, loss of balance or a righting response, reduced response to tactile stimuli 129 and slower, deeper respirations. They were held in the bag until they were sufficiently tractable or immobilized for safe handling. Typically, younger marmots (pups and yearlings) were found to be less responsive to the dosage regime than adults. The drugs also appeared to have a more profound effect on the marmots later in the active season (i.e. more pronounced in September and October). Once the marmot was sufficiently tractable, it was removed from the handling bag and further induced with inhaled isoflurane. The marmots were given a titrated gas mixture consisting of oxygen (delivered at approximately 1 liter per minute from a “click” style oxygen flow-meter / regulator) fed through a calibrated, Tech 3 isoflurane vaporizer, typically set at an initial level between 2.5 and 3.5%. The gas mixture was delivered to the animal through a modified Jackson Rees nonrebreathing circuit mated to a clear plastic anesthesia mask. The anesthesia mask was fitted with a flexible diaphragm (made from a fenestrated surgical glove taped to its rim) which formed an effective seal around the marmot’s head to minimize gas leakage. Due to the short duration of most procedures, the difficulty of visualizing the glottis and epiglottis, the potential for oropharyngeal trauma, and marmot anatomical characteristics (obligate nasal breathers and limited capacity for regurgitation and gastric reflux) marmots were not routinely intubated during anesthesia. In some instances, pups were manually restrained in the handling bag and mask induced without receiving any injectable immobilization agents. Respiratory rate, heart rate and body temperature were continuously monitored during immobilizations and anaesthesia. Respirations were monitored visually and with a pulse oximeter. Heart rate was monitored using a stethoscope, a pulse oximeter providing pulse rate data and / or a Doppler heart rate monitor with the probe positioned over the medial aspect of the distal tibia (just proximal to the tarsus). Body temperature were monitored using a digital rectal thermometer. In 130 some instances, particularly early in the active season when fat reserves are low, marmots were provided with a source of supplemental heat such as latex gloves filled with warmed water or an insulated heating pad. To effect cooling on warm days a sealed plastic bag containing flattened snow (if available) or an ice-pack were placed in the areas of greatest heat exchange (inner thigh, abdomen, shoulders, axilla) or the extremities were wetted with isopropyl alcohol. Implant surgery As much as possible, surgically implanted, intra-abdominal transmitters were used to monitor free-ranging marmots. Internal transmitters were used because the dramatic seasonal mass changes that are associated with marmot hibernation made conventional collars impractical. Implant surgeries were routinely performed in the dedicated surgery at the TBMWMRC or in the field using high standards of anesthesia and aseptic technique. Because implantation required a surgical procedure and an adequate capacity for convalescence, marmots were not implanted close to hibernation (i.e. early in the field season following emergence when body condition and food resources were low and when female marmots might be pregnant, or late in the field season close to immergence when the marmots’ metabolic rate was starting to decline and healing capacity might be reduced). Most surgeries were performed between the middle of June and the end of August. During the course of this project, temperature-responsive transmitters from Custom Telemetry ® (Watkinsville, Georgia), Telonics ® (Mesa, Arizona), Advanced Telemetry Systems ® (Isanti, Minnesota) and Holohil Systems Limited ® (Carp, Ontario) were used. The pulse rate and temperature response of the transmitters was configured to maximize functionality and prolong battery life. Pulse rate declines associated with lowered body temperature helped to prolong battery life 131 during hibernation and during the active season a low transmitter pulse rate was used to indicate potential marmot mortalities. From 1992 to 2016 a total of 898 implant surgeries were performed on Vancouver Island marmots including 96 replacement surgeries. Most of the captive marmots being prepared for release were surgically implanted with radiotransmitters and then afforded a period of convalescence in captivity. Intervals between captive surgery and release ranged from 11 to 93 days (mean = 28 days). Three marmots were held until the following year due to transient injuries that precluded same-year release and one individual died due to pre-release complications with its transmitter. 484 of the 490 captive-release marmots were surgically implanted with an abdominal radio transmitter prior to release and 32 of these individuals were later recaptured and had their transmitters replaced. One individual was recaptured a second time for transmitter replacement and one individual was recaptured and surgically implanted 50 days after being initially released without a transmitter. From 1992 to 2016 a total of 379 implant surgeries were performed on wild marmots (including marmots that were subsequently translocated), including 63 replacement surgeries. Wild marmots being intentionally prepared for translocation were surgically implanted with radio-transmitters and then allowed to convalesce at their familiar colony sites. Although 14 days was deemed to be the minimal interval between field surgery and translocation, this period actually ranged from 21 to 37 days (mean = 30 days) due to challenges associated with recapture, weather, and other field logistics. In five instances, wild marmots were not recaptured and translocated until the following year following surgery. Four marmots which were captured at aberrant locations including Bamfield, Nanaimo and Nanoose on Vancouver Island were surgically implanted and translocated to suitable habitat 132 within days of capture. All four marmots survived for at least 350 days (range 350 to 1511 days), indicating that these marmots were not adversely affected by prompt relocation following surgery. All marmots received a comprehensive physical examination prior to surgery. Individuals that were inappropriately sized, compromised by injury, or exhibiting evidence of disease were not implanted with transmitters. Surgical technique Following physical examination and placement of the monitoring equipment, marmots were positioned in a v-trough in dorsal recumbency. The fur from a small area of approximately 7 cm long × 5 cm wide was clipped from the ventral abdominal midline. The surgical site was cleaned and disinfected using a routine surgical preparation, consisting of a series of at least three chlorhexidine (or povidone iodine scrub) washes / isopropyl rinses, and finishing with a final application of a povidone iodine solution. To minimize effects on post-surgical thermoregulation, excessive removal or wetting of the fur was avoided. All surgeries were performed using sterile surgical gloves, surgical masks / caps, autoclaved, sterile surgical instruments, sterile occlusive drapes, and aseptic technique. A nonporous, transparent, self-adhesive fenestration drape (Veterinary Specialty Products, Overland Park, KS) was positioned over the surgical site. A longitudinal skin incision approximately 3 cm in length is made along the ventral midline to expose the linea alba, which was then incised after elevation of the body wall. Care was taken so that the underlying, voluminous, thin-walled intestines were not perforated during the initial incision. The incision into the abdominal cavity was extended as necessary using sharp dissection. At an appropriate point the transmitter was removed from its sterile packaging or disinfectant solution. If it had been in a disinfectant solution the unit was well rinsed with warm sterile saline or 133 lactated Ringer’s solution. The functioning and frequency of the transmitter was reconfirmed prior to placement. The transmitter was inserted into the abdominal cavity and positioned laterally and longitudinally, away from the incision site. Bleeding during the procedure was minimal. 3-0 PDS II (polydioxanone) an absorbable, synthetic, monofilament suture (Ethicon, Inc., Somerville, NJ) was used for closure of all tissue layers. Simple, interrupted sutures were used to close the body wall. A simple, continuous pattern was used to close the subcutaneous fat and a subcuticular suture was used to close the skin. Additional simple interrupted skin sutures and a thin layer of surgical tissue adhesive (Vetbond, 3M, St Paul, MN), were also be used to provide additional security of the skin incision. Beginning in 2013, injectable analgesics (Subcutaneous injection of 0.1 to 0.2 mg/kg of Meloxicam, Metacam, Boehringer Ingelheim Vetmedica, Ridgefield, CT) were administered preoperatively. Antibiotics were not routinely administered but were given on occasion at the discretion of the surgeon. Recovery Following surgery, measurements and sample collection, the isoflurane was discontinued, and the marmot was given a short interval of pure oxygen. Increased muscle tone and spontaneous postural changes (head up, rising to sternal recumbency) were typically observed within minutes of discontinuing isoflurane. After these initial signs were observed, the marmot was returned to its trap and monitored for further recovery. Once the marmot had attained normal posture and demeanour its trap was covered and placed in a quiet, thermally “neutral” area (not too hot or too cold and out of direct sunlight) and checked periodically. Induction to recovery times were approximately 45 minutes, depending upon additional manipulations (measurements, diagnostics, sampling, etc.) that were being performed. Marmots were held for a minimum of 45 to 60 minutes following 134 discontinuation of the isoflurane before being returned to their nest-box or into a familiar burrow close to their original capture site. Marmots were not transported until they could effectively maintain their balance within the trap (verified by slight tipping of the trap) in order to reduce the risk of trauma associated with losing their balance while being carried over rough terrain. Mild and transient post-operative imbalance was attributed to the residual effects of ketamine. The duration of the midazolam’s sedative effects lasted 60 to 90 minutes (exceeding ketamine’s duration of action which is approximately 30 to 45 minutes) and wild marmots typically remained calm during recovery and subsequent transport back to the release site. Potential complications associated with implantation were mitigated by minimizing animal stress and handling time, employing sound surgical techniques, and by careful selection and handling of the implant units. From 1992 to 2010, transmitters were disinfected by soaking in a povidone-iodine solution for a minimum of 12 hours. These units were subsequently rinsed with Lactated Ringers Solution or physiological saline prior to placement. Beginning in 2011, all transmitters were gas sterilized with ethylene oxide. Transmitters with surface defects were avoided, particularly in the case of “soft” encapsulation materials such as Plasti-dip® and wax. Observations during early replacement surgeries indicated that surface irregularities may have led to the development of focal adhesions between the transmitter and the greater omentum. To date, two fatal complications arising directly from an implanted transmitter have been documented. In one case, extreme post-emergence weight loss in a structurally large, captive-release adult male resulted in the transmitter becoming lodged in its pelvic canal, resulting in a fatal impaction of the gastrointestinal tract. In the second case, involving a captive marmot scheduled for release, the outer surface of the transmitter caused an extreme, chronic inflammatory reaction with significant visceral adhesions which 135 could not be surgically resolved. These problems were subsequently mitigated by having the manufacturer increase the diameter of the transmitters and by only using transmitters encapsulated in a hard, biologically inert resin. Lateral (left) and ventrodorsal radiographs of free-floating abdominal radio-transmitter in female Vancouver Island Marmot (Marmota vancouverensis). The cylindrical unit was 25 mm x 90 mm and weighed 50 grams. 136 Appendix G. Summary of morbidity & mortality in captive & free-ranging Vancouver Island Marmots (Marmota vancouverensis), 1992 to 2016 System Diagnosis Identified or diagnosed by clinical signs or physical examination Diagnosis requires necropsy or ancillary lab test(s) Identified in free-ranging marmots Identified in captive marmots abdominal abdominal abscess N Y N Y abdominal peritonitis N Y N Y abdominal colonic impaction due to transmitter N Y Y N abdominal transmitter reaction Y confirmed N Y cardiovascular atrial septal defect Y confirmed N Y cardiovascular persistent tachycardia Y confirmed N Y cardiovascular patent foramen ovale Y confirmed N Y cardiovascular cardiomyopathy Y confirmed N Y cardiovascular endocardiosis Y confirmed N Y cardiovascular cerebral hemorrhage N Y N Y cardiovascular atherlosclerosis N Y N Y dental dental malocclusions Y N N Y dental unilateral / bilateral fractures of upper incisors Y N Y Y gastrointestinal segmented enteritis, N Y N Y gastrointestinal roundworms Y/N Y Y Y gastrointestinal tapeworms Y/N Y Y Y gastrointestinal mesenteric torsion N Y N Y gastrointestinal duodenal ulcer and peritonitis N Y N Y gastrointestinal pancreatitis with peritonitis N Y N Y gastrointestinal perforated ceacal ulcer with peritonitis N Y N Y hepatic cholangiohepatitis N Y N Y hepatic hepatitis N Y N Y integumentary fleas Y N Y Y integumentary ticks Y N Y N integumentary facial abscess Y N Y Y integumentary mites Y confirmed Y Y integumentary cutaneous bots Y N N Y multisystemic stunting, post weaning un-thriftiness Y N Y Y multisystemic septicemia N Y N Y multisystemic capture-related hyperthermia, Y N Y N multisystemic facial neoplasia Y confirmed N Y multisystemic perivasculitis N Y N Y multisystemic malignant histiocytosis N Y N Y multisystemic conspecific trauma Y confirmed Y Y 137 Appendix G. (cont.) Summary of morbidity & mortality in captive & free-ranging Vancouver Island Marmots (Marmota vancouverensis), 1992 to 2016. System Diagnosis Identified or diagnosed by clinical signs or physical examination Diagnosis requires necropsy or ancillary lab test(s) Identified in free-ranging marmots Identified in captive marmots multisystemic neoplasia Y/N confirmed N Y musculoskeletal spondylosis N Y N Y musculoskeletal scoliosis Y confirmed N Y musculoskeletal hind foot aplasia Y N N Y musculoskeletal toe injuries from conspecific trauma Y N Y Y musculoskeletal chronic fracture of right olecranon Y confirmed N Y musculoskeletal fracture of carpal bones Y confirmed Y N musculoskeletal post hibernation emaciation Y confirmed Y Y musculoskeletal abdominal hernia Y confirmed N Y musculoskeletal resorption of head and neck of femur N Y N Y musculoskeletal degeneration and herniation of intervertebral disc(s) Y confirmed N Y neurologic head trauma Y confirmed N Y neurological menigoencephalomyelitis Y confirmed N Y ocular unilateral anopthalmis Y N N Y ocular bilateral, congenital cataracts Y confirmed N Y ocular corneal trauma with opacity Y confirmed Y Y ocular lenticular opacity Y N N Y ocular unilateral, corneal opacity Y N N Y ocular narrowed palpebral fissure Y N N Y respiratory bronchopneumonia Y/N confirmed N Y respiratory pulmonary adenomatosis N Y N Y respiratory laryngeal occlusion N Y N Y urogenital vaginitis, urethritis and cystitis N Y N Y urogenital paraphimosis Y N N Y 138 Appendix H. Summary of annual mortality categories in free-ranging Vancouver Island Marmots (Marmota Year 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 Total vancouverensis) 1992 to 2016. Wild 1 0 2 1 0 0 3 0 0 2 6 1 4 1 3 1 0 2 10 7 8 18 30 26 13 139 Predation Hibernation 0 0 0 0 2 0 1 0 0 0 0 0 3 0 0 0 0 0 2 0 5 1 1 0 3 1 0 1 2 0 0 0 0 0 2 0 6 0 5 0 1 2 13 1 16 1 4 4 7 2 73 8 Other Unknown 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1 0 0 0 0 0 4 0 2 0 5 0 4 0 13 1 17 0 4 3 55 Translocated 0 0 0 0 5 0 0 0 0 0 1 2 0 0 0 0 0 1 1 0 1 2 4 10 20 44 Predation Hibernation Other Unknown 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 4 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 1 0 0 0 0 0 0 0 1 0 0 0 2 0 1 0 3 0 0 0 10 0 0 0 20 5 5 0 34 Captiverelease 0 0 0 0 0 0 0 0 0 0 0 3 1 9 10 14 26 28 62 47 47 28 16 26 14 330 Predation Hibernation 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3 0 1 0 4 4 7 3 9 2 12 7 11 7 13 27 6 7 6 11 4 3 4 5 6 1 2 3 87 80 Other Unknown 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1 2 0 7 1 9 0 22 0 34 0 30 1 20 1 6 0 19 0 9 5 158 Preconditioned 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 3 10 4 20 Predation Hibernation Other Unknown 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 1 0 0 0 0 0 0 1 0 3 0 0 0 2 0 8 0 0 0 4 2 5 0 13 Total mortalities 1 0 2 1 5 0 3 0 0 2 7 6 5 10 13 15 26 31 73 54 57 49 53 72 48 533 138 139 Appendix I. A comparison of common body condition indices in wild & captive male Vancouver Island Marmots (Marmota vancouverensis). Unpublished data. Body mass divided by body length (BM/BL) Quetlet's Index Body mass divided by body length squared (B/L²) Fulton's Index Body mass divided by body length cubed (B/L³) Logtransformed body mass divided by logtransformed body length (log BM/log BL) Scaled mass index (gm) (a) Scaled neck index (cm) (b) Captive males 89.75397 1.740848 0.03404794 2.137128 4110.01 26.63 Wild males 74.59246 1.520057 0.03149986 2.101056 3816.77 24.05 t 10.384 9.7093 5.761 9.4526 5.42 14.64 df 366.411 382.125 419.318 348.421 420.23 413.15 PV < 2.2e-16 < 2.2e-16 1.62E-08 < 2.2e-16 9.90E-08 < 2.2e-16 (a) Scaled mass index (sci) comparing body mass of captive and wild marmots according to a standardized body length of 49.45 cm (mean body length for all samples). (b) Scaled neck index - comparing neck circumference of captive and wild marmots according to a standardized body length of 49.45 cm (mean body length for all samples). 139 140