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Monitoring Oral Temperature, Heart Rate, and Respiration Rate of Field-Captured Florida and Antillean Manatees (Trichech...

Permanent Link: http://ufdc.ufl.edu/UFE0021877/00001

Material Information

Title: Monitoring Oral Temperature, Heart Rate, and Respiration Rate of Field-Captured Florida and Antillean Manatees (Trichechus manatus latirostris and T. m. manatus)
Physical Description: 1 online resource (101 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: blood, heart, latirostris, mammal, manatee, manatus, marine, oral, rate, respiration, temperature, trichechus
Veterinary Medicine -- Dissertations, Academic -- UF
Genre: Veterinary Medical Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Knowledge gained from the capture of manatees for health assessment and telemetry based studies is required for the conservation of the species. Understanding how free-ranging manatees respond to capture and how to effectively monitor the awake individual animal is of the utmost concern for researchers charged with their care in the field. The main objectives of this study were as follows: 1) determine practical field methods to monitor oral temperature (OT), heart rate (HR), and respiration rate (RR) of captured manatees; 2)establish normal OT, HR, RR parameters with correlations to blood chemistry; 3) provide an easy to reference OT, HR, RR monitoring field guide for manatee researchers. Three digital thermometer types and a human bedside electrocardiogram (ECG) were tested in the field. Oral temperature measurements between three digital thermometer types showed no statistical significant difference (p=0.125). However, a thermocouple type thermometer did present several clinically significant incorrect measurements. The ECG unit was 84% successful in acquiring a heart rate during field application. Electrocardiogram HR measurements were not significantly different from those obtained by stethoscope (p=0.313). Thirty-eight Florida manatees (Trichechus manatus latirostris) were captured in Florida. Thirty-five Antillean manatees (T. m. manatus) were captured in Belize and thirteen Antillean manatees were captured from Puerto Rico. Manatees were monitored for OT, HR, and RR following capture and during handling. Blood was concurrently sampled from monitored individuals. Creatine kinase activity (CK), potassium (K+), serum amyloid A (SAA), and lactate levels were measured. Animals were removed from mean water temperatures of 25.9 C in Florida; and 28.9 C in Belize and Puerto Rico. Mean OT of Florida (n=21) and Antillean (n=26) manatees increased during capture from 32.6 C and 34.6 C; to 34.8 C and 35.2 C, respectively. There was no statistically significant difference of OT between Antillean and Florida manatees after 40 minutes (p=0.4186). Mean HR of Florida (n=25) and Antillean (n=22) manatees decreased from 66 beats per minute (bpm) and 75 bpm, respectively, to 60 bpm and 61 bpm, respectively. There was no statistically significant difference of HR between Florida and Antillean manatees after 25 minutes (p=0.0739). Mean RR of Florida (n=37) and Antillean (n=48) manatees decreased from 6 breaths/5min and 9 breaths/5 min, to 4 breaths/5 min and 5 breaths/5 min, respectively. There was no statistically significant difference in RR after 50 minutes between Florida and Antillean manatees (p=0.0943). Higher respiratory rate over time was associated with higher lactate values. Antillean manatees had higher overall lactate values than Florida manatees (p= < 0.001). Further study regarding manatee physiology is suggested to better explain the differences observed between subspecies in this study. The effect of capture on manatee OT, HR, and RR in the field shows biological significance. Monitoring of these parameters can help to improve the ability to obtain individual health assessments of manatees in the field.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis: Thesis (M.S.)--University of Florida, 2008.
Local: Adviser: Reep, Roger L.
Local: Co-adviser: Harr, Kendal Elizabeth.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-05-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0021877:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021877/00001

Material Information

Title: Monitoring Oral Temperature, Heart Rate, and Respiration Rate of Field-Captured Florida and Antillean Manatees (Trichechus manatus latirostris and T. m. manatus)
Physical Description: 1 online resource (101 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: blood, heart, latirostris, mammal, manatee, manatus, marine, oral, rate, respiration, temperature, trichechus
Veterinary Medicine -- Dissertations, Academic -- UF
Genre: Veterinary Medical Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Knowledge gained from the capture of manatees for health assessment and telemetry based studies is required for the conservation of the species. Understanding how free-ranging manatees respond to capture and how to effectively monitor the awake individual animal is of the utmost concern for researchers charged with their care in the field. The main objectives of this study were as follows: 1) determine practical field methods to monitor oral temperature (OT), heart rate (HR), and respiration rate (RR) of captured manatees; 2)establish normal OT, HR, RR parameters with correlations to blood chemistry; 3) provide an easy to reference OT, HR, RR monitoring field guide for manatee researchers. Three digital thermometer types and a human bedside electrocardiogram (ECG) were tested in the field. Oral temperature measurements between three digital thermometer types showed no statistical significant difference (p=0.125). However, a thermocouple type thermometer did present several clinically significant incorrect measurements. The ECG unit was 84% successful in acquiring a heart rate during field application. Electrocardiogram HR measurements were not significantly different from those obtained by stethoscope (p=0.313). Thirty-eight Florida manatees (Trichechus manatus latirostris) were captured in Florida. Thirty-five Antillean manatees (T. m. manatus) were captured in Belize and thirteen Antillean manatees were captured from Puerto Rico. Manatees were monitored for OT, HR, and RR following capture and during handling. Blood was concurrently sampled from monitored individuals. Creatine kinase activity (CK), potassium (K+), serum amyloid A (SAA), and lactate levels were measured. Animals were removed from mean water temperatures of 25.9 C in Florida; and 28.9 C in Belize and Puerto Rico. Mean OT of Florida (n=21) and Antillean (n=26) manatees increased during capture from 32.6 C and 34.6 C; to 34.8 C and 35.2 C, respectively. There was no statistically significant difference of OT between Antillean and Florida manatees after 40 minutes (p=0.4186). Mean HR of Florida (n=25) and Antillean (n=22) manatees decreased from 66 beats per minute (bpm) and 75 bpm, respectively, to 60 bpm and 61 bpm, respectively. There was no statistically significant difference of HR between Florida and Antillean manatees after 25 minutes (p=0.0739). Mean RR of Florida (n=37) and Antillean (n=48) manatees decreased from 6 breaths/5min and 9 breaths/5 min, to 4 breaths/5 min and 5 breaths/5 min, respectively. There was no statistically significant difference in RR after 50 minutes between Florida and Antillean manatees (p=0.0943). Higher respiratory rate over time was associated with higher lactate values. Antillean manatees had higher overall lactate values than Florida manatees (p= < 0.001). Further study regarding manatee physiology is suggested to better explain the differences observed between subspecies in this study. The effect of capture on manatee OT, HR, and RR in the field shows biological significance. Monitoring of these parameters can help to improve the ability to obtain individual health assessments of manatees in the field.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis: Thesis (M.S.)--University of Florida, 2008.
Local: Adviser: Reep, Roger L.
Local: Co-adviser: Harr, Kendal Elizabeth.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-05-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0021877:00001


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1 MONITORING ORAL TEMPERATURE HEART RATE, AND RESPIRATION RATE OF FIELD-CAPTURED FLORIDA AND ANTILLEAN MANATEES ( Trichechus manatus latirostris and T. m. manatus ) By ARTHUR WILLIAM WONG A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2008

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2 2008 Arthur William Wong

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3 To my parents: Bill and Alice. Thank you for everything.

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4 ACKNOWLEDGMENTS The com pletion of this thesis was made possible with the support of many scientists, veterinarians, students, friends, and family. It wa s from their collaborative effort that I was able to meet the challenges of graduate school while working with the Florida Fish and Wildlife Conservation Commission. I am grateful for the educational and professi onal opportunities that I have had during the time of this study. I am especially thankful to Bob Bonde, w ho guided me during every field capture and throughout many aspects of this study. I tha nk him for his tutelage, unwavering support, kindness, humor, and friendship. I want to than k his wife and fellow biologist Cathy Beck, and his children Mike, and Julie for warmLy opening their home to me for so many years. I would also like to thank my committee co-chair Dr. Kendal Harr whose steadfast determination, intellect, and advisement were instrumental in the construction of th is thesis. I am thankful to my other committee co-chair Dr. Roger Reep fo r his professional oversight and wisdom during the academic process. I thank Dr. Elsa Haubol d for her professional support and vision in realizing the benefits of this study from a manage ment perspective. I am very grateful to Dr. Andy Stamper whose patience, thoughtfulness, and knowledge were critical toward the quality of the research. I give a heart felt thanks to Dr. Ruth Francis-Floyd who accepted me early on as a student, thereby providing me with an educational opportunity that has changed my life forever. I have to thank students and staff of the Univer sity of Florida. Thanks to James Colee for his statistical advisement. I am very thankful to Melanie Pate fo r all her hard work in the lab, and her great sense of humor when working with me. Thanks to Dr. John Harvey for allowing me to use his lab space whenever I needed. A whole hearted thanks to Dr. Jessica-Siegal Willott whose friendship and advice helped me to get through many difficult times. Thank you to Katie Tripp for her willingness to immediately help me with data requests on often short notice, while

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5 still working on her dissertation. A very special thanks to Sally OConnell for always informing me of my academic deadlines, and properly registering me for classes. Id like to thank Dr. Charlie Courtney, for taking the time to talk wi th me about my professional development and providing me with the tuition support even when this study took longer than expected. I offer thanks to all my colleagues at the Florida Fish and Wildlife Conservation Commission. I would like to especially thank Jamison Smith for his encouragement and support as a supervisor when I proposed the idea for this study and still wo rk with the agency. I also thank him and his wife Amy for their great friend ship. Thanks to Alicia Windham-Reid for her support during often hectic days of work. Tha nks to Dr. Allen Foley for answering my many research questions. Thanks to Dr. Rommel Sen tiel for answering my questions and providing his support for my admission to graduate school. I am thankful to J udith Buhrman for her introspective conversations and providing me with the many journal articles I requested. I am thankful to Dr. Chip Deutsch and Dr. Holly Edwards for kindly accommodating me during field captures in Tampa, Florida. Thanks to vol unteers Janice Price and Mindy Kirkland for their extra work supporting this study. Thanks are given to all the staff of the USGS-S irenia Project. Especially, I thank Jim Reid for his patience, kindness, and friendship. I thank him for enthusia stically teaching and supporting me during captures in Na ples, Florida and Puerto Rico. Thanks to Susan Butler for always ensuring that I was well taken care of in the field. Thanks are also given to all th e staff of Wildlife Trust. A special thanks to James Buddy Powell for his patience, understanding, and suppor t while conducting captures in Belize. I am grateful that he allowed me to explore and learn in such an exciting, and fun environment. I am

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6 also thankful to his wife Maur een and daughter Morgan for their cheerful company in the field. Thanks to Nicole Auil for accommodating my research needs. Additionally, thanks to the U.S. Fish and W ildlife Service in Puerto Rico for supporting my research needs. Thanks to Dr. Tony Mignucci for opening his home to me in Puerto Rico. Thanks to Ben Winton of Florida Medical Sa les and his daughter Megan for providing the electrocardiogram unit used in this study. Tha nks to Dr. David Murphy and the staff of the Parker Bradenton Aquarium for allowing us to work with Snooty the manatee. Thanks to Nancy Hines and her son Isaiah for providing warm meals and rooms at Manatee Lodge in Belize. Thanks to Angela Gall and Yvonne Dartsch for their extra assistance in Belize. I thank my dear friend Jesse Galsim for the many la te nights he donated toward working on the monitoring field guide. And thanks to his very understanding wife Linda for putting up with my constant phone calls. Finally, I would like to thank my entire hila rious family and all my super friends for always reminding me to keep things in perspec tive. Special thanks to my sister Karyn and brother Mike for their candid sibli ng commentary during this process.

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7 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........9 LIST OF FIGURES.......................................................................................................................11 ABSTRACT...................................................................................................................................12 CHAP TER 1 INTRODUCTION..................................................................................................................14 2 METHODS DEVELOPMENT FOR FI ELD MONITORING OF MANATEE ( Trichechus manatus latirostris, T. m. manatus ) ORAL TEMPERATURE AND HEART RATE........................................................................................................................17 Abstract....................................................................................................................... ............17 Introduction................................................................................................................... ..........17 Materials and Methods...........................................................................................................18 Manatees Sampled...........................................................................................................18 Oral Temperature Method and Thermometer Validation................................................ 18 Heart Rate Monitoring and ECG validation.................................................................... 19 Statistics...........................................................................................................................21 Results.....................................................................................................................................22 Oral Temperature Method a ndThermom eter Validation................................................. 22 Heart Rate Monitoring and ECG validation.................................................................... 23 Discussion...............................................................................................................................23 Oral Temperature Method and Thermometer Validation................................................ 23 Heart Rate Monitoring and ECG Validation................................................................... 25 Future Considerations: Oral Temper ature and H eart Rate Monitoring........................... 28 3 ORAL TEMPERATURE, HEART RATE, AND RESPIR ATION RATE OF MANATEES ( Trichechus manatus latirostris, T. m. manatus ) EXPOSED TO CAPTURE AND HANDLI NG IN THE FIELD.................................................................... 33 Abstract....................................................................................................................... ............33 Introduction................................................................................................................... ..........34 Materials and Methods...........................................................................................................35 Blood Sampling and Biochemical Analysis.................................................................... 38 Statistical Analyses.......................................................................................................... 39 Results.....................................................................................................................................40 Capture............................................................................................................................40 Oral Temperature.............................................................................................................41 Heart Rate..................................................................................................................... ...42

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8 Respiration Rate.............................................................................................................. 42 Blood Chemistry: Creatine Kinase, Pota ssium Serum Amyloid A, and Lactate........... 43 Respiration Rate and Lactate........................................................................................... 44 Other Significant Findings.............................................................................................. 44 Discussion...............................................................................................................................45 Capture............................................................................................................................45 Oral Temperature.............................................................................................................45 Heart Rate..................................................................................................................... ...46 Respiration Rate.............................................................................................................. 47 Blood Chemistry: Creatine Kinase, Pota ssium Serum Amyloid A, and Lactate........... 48 Respiration rate and lactate............................................................................................. 50 Florida and Antillean Manatees: Differences in Oral Tem perature; Heart Rate, Respiration Rate, and Lactate...................................................................................... 51 Oral Temperature.....................................................................................................51 Heart Rate, Respirati on Rate, and Lactate ............................................................... 51 Abnormal Individuals...................................................................................................... 52 Future Monitoring Considerations..................................................................................53 4 MANATEE TEMPERATURE, HEART, AND RESPIRATI ON MONITORING FIELD GUI DE.................................................................................................................... ....62 5 CONCLUSION..................................................................................................................... ..63 Hypotheses Addressed........................................................................................................... .63 The Captured Manatee and Future Monitoring Considerations.............................................64 APPENDIX A CAPTURE LOCATIONS...................................................................................................... 68 B BASIC MANATEE CAPTURE METHOD PHOTOS.......................................................... 70 C MANATEE LENGTH VE RSUS WEIGHT ........................................................................... 74 D BASIC ECG RESULTS.........................................................................................................75 E PLASMA CLINICAL BIOCHEMISTRY AND PROGESTERONE 4 VALUES ................ 76 F MANATEE MONITORING FIELD GUIDE........................................................................ 84 LIST OF REFERENCES...............................................................................................................96 BIOGRAPHICAL SKETCH.......................................................................................................101

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9 LIST OF TABLES Table page 2-1 Results of 0C water bath measuremen ts by Radioshack (RS) and Digisense therm ocouple (DS) thermometers...................................................................................... 29 2-2 Results of 15C water bath measuremen ts by Ra dioshack (RS) and Digisense thermocouple (DS) thermometers...................................................................................... 29 2-3 Results of 30C water bath measuremen ts by Ra dioshack (RS) and Digisense thermocouple (DS) thermometers...................................................................................... 29 2-4 Results of 45C water bath measuremen ts by Ra dioshack (RS) and Digisense thermocouple (DS) thermometers...................................................................................... 30 2-5 Results of 60C water bath measuremen ts by Ra dioshack (RS) and Digisense thermocouple (DS) thermometers...................................................................................... 30 2-6 Results of 75C water bath measurem ents by Di gisense thermocouple (DS) thermometer.................................................................................................................... ...30 2-7 Evaluation of ECG perf orm ance on 25 manatees:............................................................. 31 3-1 Apparently healthy, free-ranging manat ees m onitored during captures from 2004 to 2006....................................................................................................................................55 3-2 Air and water temperatures (C) associated with manatees sampled for oral temperature, heart rate and respiration rate.......................................................................55 3-3 Descriptive statistics for oral temperat ures of Florida and Antillean m anatees at different time intervals during field monitoring................................................................ 57 3-4 Descriptive statistics for heart rates of Florida an d Antillean manatees at different time intervals during field monitoring............................................................................... 58 3-5 Descriptive statistics for respiration rates of Florida and Antillean m anatees at different time intervals during field monitoring................................................................ 59 3-6 Initial heart rate respiration rate and lactate of a fem ale Antillean manatee BZ97F03, captured on 3 separate occasions during this study...........................................................61 3-7 Mean times in minutes during capture and handling of m anatees..................................... 61 A-1 Associated location descriptions and coordinates for Figure A-1 ..................................... 69 D-1 Results of ECG printed QRS tracing an alyses for 25 of 47 heart rate m onitored manatees....................................................................................................................... ......75

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10 E-1 Individual plasma clinical biochem istry of 38 Florida (Trichechus manatus latirostrus ) and 48 Antillean (T. m. manatus ) manatees concurrently monitored for oral temperature, heart ra te, and respiration rate............................................................... 76 E-2 Plasma clinical biochemistry descriptive statistics from 38 Florida ( Trichechus manatus latirostrus ) and 48 Antillean (T. m. manatus ) fieldcaptured manatees............. 80 E-3 Abnormal creatine kinase (CK) activity identified from 38 Florida (Trichechus manatus latirostrus ) and 48 Antillean (T. m. manatus ) fieldcaptured manatees............. 81 E-4 Abnormal lactate values identified from 38 Florida ( Trichechus manatus latirostrus ) and 48 Antillean ( T. m. manatus ) fieldcaptured m anatees.............................................. 81 E-5 Abnormal serum amyloid A (SAA) va lues identified from 38 Florida ( Trichechus manatus latirostrus ) and 48 Antillean (T. m. manatus ) fieldcaptured manatees............. 81 E-6 Abnormal potassium (K+) values identified from 38 Florida ( Trichechus manatus latirostrus ) and 48 Antillean (T. m. manatus ) fieldcaptured manatees........................... 82 E-7 Progesterone 4 values from blood serum of heart rate m onitored female manatees, determined by chemiluminescent immunoassay (Tripp et al. 2006).................................83

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11 LIST OF FIGURES Figure page 3-1 OT, HR, and RR values in relation to invasive procedures that occurred to BZ05M79, a 225 cm male, on April 17, 2005................................................................... 56 3-2 Mean oral temperatures over time, with st andard error bars, f or healthy juvenile and adult manatees, collected immediately following capture................................................. 57 3-3 Mean heart rates over time, with standard error bars, for healthy juvenile and adult m anatees, immediatel y following capture......................................................................... 58 3-4 Mean respiration rates over tim e, with standard error ba rs, for healthy juvenile and adult manatees, immediately following capture................................................................ 59 3-5 Correlation analyses of aver age h eart rate versus averag e respiration for Florida and Antillean manatees.............................................................................................................60 A-1 Map of manatee capture locations..................................................................................... 68 B-1 Large seine net deployed from a capture boat................................................................... 70 B-2 Large seine net enclosed on a manatee.............................................................................. 71 B-3 Manatee lifted onto a capture boat..................................................................................... 71 B-4 Land set near Floridas Teco Power Plant. In this m anner, multiple manatees would be captured.........................................................................................................................72 B-5 A manatee capture in Belize.............................................................................................. 73 C-1 Length weight relationshi p of m anatees captured in this study, from Florida and Belize......................................................................................................................... ........74

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12 Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science MONITORING ORAL TEMPERATURE, HE ART RATE, AND RESPIRATION RATE OF FIELD CAPTURED FLORIDA AND ANTILLEAN MANATEES ( Trichechus manatus latirostris and T. m. manatus ) By Arthur William Wong May 2008 Chair: Roger Reep Cochair: Kendal Harr Major: Veterinary Medical Sciences Knowledge gained from the captu re of manatees for health assessment and telemetry based studies is required for the conservation of the species. Understanding how free-ranging manatees respond to capture and how to effectively monitor the awake individual animal is of the utmost concern for researchers charged with their care in the field. The main objectives of this study were to: 1) determine practical field methods to monitor oral temperature (OT), heart rate (HR), and respiration rate (RR) of captured manatees; 2) establish normal OT, HR, RR parameters with correlations to blood chemistry; 3) provide an ea sy to reference OT, HR, RR monitoring field guide for manatee researchers. Three digi tal thermometer types and a human bedside electrocardiogram (ECG) were tested in the fiel d. Oral temperature measurements between three digital thermometer types showed no statistical significant difference ( p =0.125). However, a thermocouple type thermometer did present several clinically significant incorrect measurements. The ECG unit was 84% successf ul in acquiring a heart rate during field application. Electrocardiogram HR measurements were not significantly different from those obtained by stethoscope ( p=0.313). Thirty-eight Florida manatees ( Trichechus manatus latirostris ) were captured in Florida. Thirty-five Antillean manatees ( T. m. manatus ) were

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13 captured in Belize and thirteen Antillean manatees were captured from Puerto Rico. Manatees were monitored for OT, HR, and RR follo wing capture and during handling. Blood was concurrently sampled from monitored individual s. Creatine kinase activity (CK), potassium (K+), serum amyloid A (SAA), and lactate levels were measured. Animals were removed from mean water temperatures of 25.9 (.2) C in Fl orida; and 28.9 (.5) C in Belize and Puerto Rico. Mean OT of Florida (n=21) and Antillean (n=26) manatees increased during capture from 32.6 (.8) C and 34.6 (.9) C; to 34.8 (.5) C and 35.2 (.7) C, respectively. There was no statistically significant differe nce of OT between Antillean and Florida manatees after 40 minutes ( p=0.4186). Mean HR of Florida (n=25) and An tillean (n=22) manatees decreased from 66 (.7) beats per minute (bpm) and 75 (.7) bpm, respectively, to 60 (.9) bpm and 61 (.5) bpm, respectively. There was no statistic ally significant difference of HR between Florida and Antillean manatees after 25 minutes (p=0.0739). Mean RR of Florida (n=37) and Antillean (n=48) manatees decreased from 6 (.9) breaths/5min and 9 (.5) breaths/5min, to 4 (.0) breaths/5min and 5 (.3) breaths/5min, respectively. There was no statistically significant difference in RR after 50 minutes between Florida and Antillean manatees ( p=0.0943). Higher respiratory rate over time was associated with higher lactate values. Antillean manatees had higher overall lactate values than Florida manatees ( p=<0.001). Further study regarding manatee physiology is suggested to better explain the differences observed between subspecies in this study. The effect of capture on manatee OT, HR, and RR in the field shows biological significance. Mo nitoring of these parameters can help to improve the ability to obtain individual health assessmen ts of manatees in the field.

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14 CHAPTER 1 INTRODUCTION The Florida m anatee, Trichechus manatus latirostris, is classified as an endangered species in the State of Florida, protected by the U. S. Endangered Species Act, and by the Marine Mammal Protection Act, listed in the Conventio n on International Trade of Endangered Species of Wild Flora and Fauna under Appendix I, and de fined as vulnerable by the International Union for Conservation of Nature and Natural Res ources (U.S. Fish and Wildlife Service 2004, Convention on International Trade of Endange red Species of Wild Flora and Fauna 2004, Haubold et al 2006, International Union for Conservati on of Nature and Natural Resources 2007). Given its status, multiple federal, state, private, and non-profit organizations have been working together to protect and conserve the sp ecies from anthropogenic and natural threats to its population. Long term tracking studies, population studies, rescues, and rehabilitation of manatees, all involve regular capture and handling of free-ranging individuals. During these interactions, scientists attempt to assess the overall health of the manatee. An animals overall appearance including girth, sk in condition, and semi-quantitative dorsal subcutaneous fat thickness is examined (Ward-Ge iger 1997). This assessment is a superficial method to assess external integument and body c ondition of an individual manatee, and is performed regularly by biologists. However, the evaluation of the health of a manatee is typically performed by select veterinarians or more experienced biologists. In this assessment, respiration, heart rate, body temper ature, and blood chemistry are often recorded. Protocols for monitoring these vital signs are well defined in humans and in many species of domestic and wild animals, but protocols do not exist for mana tees. Methods used to evaluate temperature, heart rate, and respiratory rate vary between reha bilitation facilities. While literature exists on manatee blood chemistry (White et al 1976, Medway et al. 1982, Walsh & Bossart 1999,

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15 Bossart et a l. 2001, Manire et al 2003, Murphy 2003), study methods are changing due to advances in medical technologies. Furthermore, correlations in body temperature, heart rate, and respiration with manatee blood chemistry and the overall health of the an imal have not been studied in a field setting. Since the late 1970s, only six manatees have died in over 1000 field research captures (U.S. Geological Survey, Florida Fish and Wild life Conservation Commission, Wildlife Trust, unpublished data). However, monitoring of or al temperature (OT), heart rate (HR), and respiration rate (RR) of capture d manatees can provide additional information that may be useful to improve the care of individuals and increa se our understanding of manatee physiology. This thesis: 1) Determines practical field me thods that can be performed by researchers in order to monitor oral temperatur e (OT), heart rate (HR), and resp iration (RR) as a basic health assessment in the manatee following capture and during handli ng. 2) Establishes a normal range of OT, HR, and RR in relation to blood chemistry values. 3) Pr ovides a manatee monitoring field guide to aid researchers during health assessments. To help accomplish the aforementioned objectives, several aims were defined, and associat ed hypotheses were proposed to be tested: Specific Aim 1: Establish temperature, heart rate, a nd respiration rate parameters in captured manatees. Hypothesis 1a : Oral temperature will show a time dependent increase when ambient air temperature is above 26C. Hypothesis 1b: A healthy captured manatee's heart rate is higher than 40 beats/minute. Hypothesis 1c : Healthy captured manatees will show a normal respiration rate of 3-4 breaths/5 minute cycle (Bossart 2001). Specific Aim 2: Determine possible correlations of OT, HR, RR with blood chemistry. Hypothesis 2a : Oral temperature will show a time dependent increase in value after capture; heart rate and respirati on rate will stabilize over time.

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16 Hypothesis 2b: A positive correlation exists betw een heart rate, potassium, and lactate concentration amongst hea lthy captured manatees. This information is novel and important as th e existing literature re flects information on manatees under captive care and not free-ranging manatees during capture. Additionally, there are no published studies on simultaneous OT, HR, and RR in field captured manatees. This thesis is organized with the intent to publish the informa tion in peer reviewed journals as a contribution to the scient ific community. The second chapter describes assessment and quality control of techniques chosen to measure OT, HR, and RR. The third chapter is an article presenting and discussing the physiological results of the study. The fourth chapter references a practical monitoring field guide located in Appendix F, which is intended for manatee researchers, and presented in book publication form at. The fifth and final chapter provides an overall thesis conclusion.

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17 CHAPTER 2 METHODS DEVELOPMENT FOR FI ELD MONITORING OF MANATEE ( Trichechus manatus latirostris, T. m. manatus ) ORAL TEMPERATURE AND HEART RATE Abstract Prior to this study, continuous m onitoring of oral temperature and heart rate was not regularly performed during health asse ssments of free-ranging manatees ( Trichechus manatus latirostris, T. m. manatus ). Three digital thermometers, an electrocardiogram (ECG), and stethoscope were tested for efficacy in manat ee field health assessments. While there was no statistically significant difference between ther mometer types in measuring oral temperature ( p=0.125), the thermocouple thermometer still pr esented several skewed oral temperature readings in the field which were clinically si gnificant, falsely indica ting hyperthermia, in a normothermic manatee. No statistically si gnificant difference was found between ECG and stethoscope heart rate measurements ( p=0.313). The ECG was 84% successful in measuring heart rate and provided continuous cardiac monito ring in a field capture setting. The most common challenge was water and sand fouling electrode connections. The ECG, stethoscope, and basic digital thermometer are recommended as tools to monitor the heart rate, and oral temperature of manatees in the field. Introduction It is stand ard practice in many medical fields to monitor temp erature, heart and respiration rate as part of regular health assessment (George 1965, Osofsky 1997) While respiration rate (RR) can be assessed using human senses alone, monitoring oral te mperature (OT) and heart rate (HR) requires instrumentation. Electronic monito ring equipment is generally developed for use in clinical settings where environmental factors, such as extreme temperature, humidity, and dirt exposure are limited. These cannot always be co ntrolled in a field se tting and can render equipment inoperable. Furthermore, many devices are designed for human or domestic animal

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18 use, and may not be useful in manatees due to anatomic and physiologi c variability, e.g. large size and thick dermal layers. Reliable monitoring equipment and implementati on is necessary to ensure proper care of manatees during field captures. In order to accomplish this goal, this study assessed the efficacy of three digital thermometers, a nd an ECG for monitoring oral temp erature and heart rate in the Florida manatee ( Trichechus manatus latirostris), and Antillean manatee ( T. m. manatus), without chemical immobilization and under li mited human restraint in the field. Materials and Methods Manatees Sampled Captures were perform ed under U.S. Fi sh and Wildlife Service permits MA773494, MA791721-4, and the Belizean Department of Forestry permit CD/60/3/05(36) for trackingbased population studies, conducted by the Fl orida Fish and Wildlife Conservation Commissions Fish and Wildlife Research Institute the U.S. Geological Survey Sirenia Project, and Wildlife Trust, respectively. Manatees in th is study were concurrently monitored for oral temperature (OT), heart rate (HR) respiration rate (RR) and sa mpled for research purposes as well as for complete health assessment. Oral Temperature Method and Thermometer Validation A Digisense (DS) thermocouple therm ometer (Cole-Parmer, Vernon Hills, IL) equipped with type T, copper-constantin thermocouple probes, coated in fl exible blue plastic (Physitemp Instruments Inc., Clifton, NJ), a Radiosh ack (RS) thermometer model 63-1009A, and a RS thermometer model 63-1035 (Radio Shack, Fort Worth, TX) were used to measure oral temperature. The RS 63-1035 continuously disp layed ambient air and oral temperatures simultaneously. The manufacturers specified acc uracy of the DS thermocouple thermometer is +/0.4C for temperatures greater than -150C. A useful range is specified by the

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19 manufacturer at 0 to 40C. The manufacturers specified a ccuracy of the RS thermometer model 63-1009A and model 63-1035 is +/1C, at 0 to 40C. To test manufacturer claims of accuracy a nd precision, each thermometer was subjected to ten rounds of a series of different temperature water baths in a cont rolled lab setting. The temperature probes were randomLy pl aced in water temperatures of 0C (ice bath with distilled water), 15C (Fisher Scientific Isotemp Refrigerated Circulator Model No. 9100 Pittsburgh, PA), 30C (Dubnoff Metabolic Shaking Incubator), 45C (Fisher Scientific 20 Liter Water Bath, Pittsburgh, PA), and 60C (Fisher Scientific 20 Liter Water Bath, Pittsburgh, PA). Additionally, only the DS thermocouple thermometer probe was tested at 75C (Sybrone Corp., Thermolyne Model No.SP-A1025B Dubuque, IA). The thermomete rs were allowed to adjust for a minimum of one minute at each temperature before a readi ng was recorded. Accurate temperatures in the baths were maintained using calibrated mercur y thermometers, considered to be the gold standard. For field use, the RS 63-1035 and DS thermo couple thermometer were each placed in a modified dry bag (West Marine, Watsonville, CA) or Ziploc bag to prevent water damage. To determine field accuracy, probe tips from the tw o thermometer types were taped together to ensure simultaneous measurements of OT from the same location. OT probes were placed once the manatee was secured on land or on a boat deck. The soft wire temperature probe was placed by hand, towards the back of the mouth, adjacent to the inferior dental mandibular cavity, located buccally past the molars. The temperature probes were removed just prior to release. Heart Rate Monitoring and ECG Validation Heart rate w as ausculted with a Sonocardia wa ter proof stethoscope (M agnafortis, Seattle, WA). The stethoscope bell was inserted undernea th the manatees axilla and placed ventrally close to midline. The number of heart beats, de fined as atrial and ventri cular contractions, were

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20 counted for 15 seconds and multiplied by four to determine beats per minute (bpm). The resulting stethoscope measurements were consid ered the gold standard when compared to ECG measurements. Prior to use in the field a Mac 500 ECG (GE He althcare, Milwaukee, WI) was tested using a four electrode placement technique on a 280 cm straight-line length, 540 kg, healthy, sexually mature, male, captive manatee at the Parker Ma natee Aquarium in Bradenton, Florida. Four commercially available adhesive LLEBL Electrode Series electrodes (L ead-Lok, Inc., Sandpoint, ID) were placed on the manatee. Two anterior leads were placed dorsally on the right and left side, approximately 2-7 cm cranial to the pectoral flipper, anterior to the scapula. Two caudal leads were placed laterally approxim ately 10 cm caudal to the pectoral flippers at th e level of the manatees shoulder (Siegal-Willott et al 2006). This lead placement was the standard used throughout this study. In the field, algae or dirt on the skin at the lead placement sites were first cleaned. The skin area was wiped with an alcohol pad, and dried with a clea n paper towel to ensure proper adherence of sticky electrode pads. Occasiona lly, a small amount of tissue glue was used directly on the cleaned site to improve electrode pad adhe rence. A drop of Aquasonic ) 100 ultra sound transmission gel (Parker laboratories, Fairfield, NJ) was plac ed at the connection point of the lead clip and the electrode pad to improve electrical conductivity. Following field exposure, lead connection po ints were wiped down with alcohol gauze pads to prevent oxidation. Lead clips were taken apart, soaked in a small jar of alcohol, and carefully brushed down with a small piece of sturdy paper towel, to rid them of sand, rust, and salt.

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21 Electrocardiogram HR was compared with simultaneous measurements obtained by stethoscope to ensure its accuracy. Printed electr ocardiograms were examined to ensure validity of the units displayed HR and to assess heart function (Siegal-Willott et al 2006). Electrocardiograms that showed artifacts such as distorted baselines, or unclear PQRS complexes were considered as evidence for an inaccurate HR reading. Data from in accurate tracings were not used and these were considered equipment failures. Based on its smaller size and simpler interface, the Mac 500 ECG unit was selected for field use. The unit was placed in a clear dry bag (West Marine, Watsonville, CA) to prevent water damage. To determine if ECG and stethos cope readings were significantly different, a paired t-test was performed comparing 56 randomLy sampled HRs, simultaneously measured by the two instruments, from 20 mana tees selected at random. To de termine the success rate of the ECG while being used in the field, successful and failed attempts to collect HRs were documented during HR monitoring of 25 manatees selected at random. A successful ECG HR measurement was determined by a steady HR acco mpanied by a readable electrocardiogram. ECG HR was checked by stethoscope for clinical accuracy. A percentage of success and error was then calculated. Statistics Basic statistical analyses were conducted us ing Microsoft Offi ce Excel 2003 for Windows (Microsoft Corp., Redmond, WA), and Sigma Stat for Windows, Version 3.1 (Systat Software Inc., Point Richmond, CA). T-tests were conducted at a significance level (Type I error rate) of = 0.05, whereby, the null hypothesis was define d as the two groups lacking significant difference. To evaluate diffe rences in field OT measurem ents between RS 63-1035 and DS thermocouple thermometers, a paired t-test was performed comparing 109 randomLy sampled OTs, simultaneously measured by the two in struments, from 11 w ild caught manatees.

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22 Results Oral Temperature Method andThermometer Validation Under laboratory testing, one DS therm oc ouple thermometer with two probes, one RS 1009A, and four RS 1035 thermometers performe d within their respec tive manufacturers specified levels of accuracy in the 0 and 30C wa ter baths (Table 2-1, 2-3). However, at the other temperature water baths, the digital thermo meters showed several readings that were less accurate, with the DS thermocouple thermomete r showing the highest range of variability outside of the 0 to 40C useful range (Table 22,Table 2-4 through Table 2-6). This was most evident when the DS thermometer presented a re ading of 33.5C after a mi nute in the 45C water bath. In the 15C and 60C water baths the RS 1009A and DS thermocouple showed several inaccurate readings, but mean temperature re adings were not significantly affected. A grand total of 109 randomLy selected simultane ous oral temperature readings were taken from 9 Florida manatees, 2 Belize manatees using the RS and DS thermometers. The RS 1035 mean temperature was 34.5 (.2) C and the DS thermocouple mean temperature was 34.3 (.1) C. A paired t-test of the grand total resulte d in no statistically significant difference between the oral temperature readin gs of the two thermometer types (p=0.125). However, a paired t-test of 12 consecutive temperatures gathered from one individual resulted in a statistically significant difference between th e measurements of the two thermometers ( p<0.001). In this instance, the RS 1035 mean temperat ure was 35.7 (.2) C and the DS thermocouple mean temperature was 34.0 (.9) C. The bigge st measurement discrepancy from this data sampling session was an RS 1035 measurement of 35.6C and a DS thermocouple measurement of 32.5C.

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23 Heart Rate Monitoring and ECG Validation A paired t-test of 56 random Ly selected simultaneous ECG and stethoscope measurements from 10 Florida manatees and 13 Antillean manatees resulted in no significant difference between the two instruments values ( p = 0.313). Mean HR via ECG was 58 bpm () and mean HR via stethoscope was 60 bpm (). Howeve r, several clinically significant differences between ECG HR and stethoscope HR were appa rent from three Florida manatees. Whereby, combined ECG HR values were 56, 53, 38, 46, 60 bp m; and simultaneous stethoscope HR values were 32, 44, 48, 61, and 80 bpm respectively. A total of 116 ECG readings were attempted on 11 Florida manatees and 14 Antillean manatees (Table 2-7). The ECG was 83.6% succ essful in obtaining HRs and paper tracings in the field; 9.5% of the unsuccessful tracings were associated with poor lead setup such as wet or dirty electrode clips and 6.9% were associated with printer problems. Discussion This study provides a m inimally invasive and e ffective OT and HR monitoring protocol for manatees using commercially available instrumentation. Oral temperature is effectively measured using a basic indoor/outdoor thermo meter. The ECG monitoring as described by Siegal-Willott et al (2006) produces reliable results that can effectivel y be repeated by field researchers. Furthermore, the resulting inform ation from this study provides useful detailed information as to the challenge s faced when attempting to con tinuously monitor OT and HR of free ranging manatees. Oral Temperature Method and Thermometer Validation Oral tem perature measurement has been performe d in a captive setting on manatees and its reported normal range of 35.5C to 36C is reflective of the reported normal core body temperature range of 35.6C to 36.4C (Gallivan et al 1983, Irvine 1983, Murphy 2003). The

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24 placement of a soft wire temperature probe orally is considerably easier than attempting to place a probe rectally to obtain core body temperature, as has been performed on many large animal species such as dolphins, el ephants, and moose (Sweeney & Ridgway 1975, Osofsky 1997, Franzmann et al 1984). Rectal temperature probe placem ent is not practiced on manatees as they are most safely secured in a ventrally r ecumbent position thereby bl ocking rectal access. Should rectal access even be obtained, the reported range of 27C to 32C is significantly less than core body temperature, and may be clinical ly less significant (Irv ine 1983). Furthermore, fecal build up in the manatee colon may affect temperature readings, as has been demonstrated with elephants (Buss & Wallner 1965). Typically for free rangi ng ungulates, rectal temperature is only taken on chemically immobilized individuals. For free-ranging dolphins, human restraint is generally adequate for rectal access. Howe ver, physical restraint of large, powerful, and awake manatee is dangerous. The DS thermocouple thermometer under laborato ry and field testing showed the highest variability in measurements. While the DS thermocouple thermometer can function in a wide range of temperatures, this is not necessary for the narrow biologica l range of manatee body temperature. The DS thermocouple thermome ter has two connection points to allow for simultaneous measurements with many different probe types. This function adds to the versatility of the unit, but may also add to environmental exposure of electronic elements, which could affect measurement accuracy. The outlier m easurements seen in the field are possibly due to comparably less ideal environmental conditions such as salt water, humidity, sand, and heat. In contrast the RS 63-1035 and 63-1009A are much simpler units, with no option to calibrate, and no probe connection points. With fewer parts, these thermometers may have less of a chance for malfunction and error. Assessing accuracy of thermometers in a laboratory setting provides

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25 an insight to performance potential in the fi eld. Under such ideal conditions individual measurements outside the predicted scope of accu racy should be given careful consideration as dirt, water, and salt spray can seriously damage electrical equipment. Regular temperature bath testing of electronic thermometers should be perf ormed in order to ensure accuracy over time, using a mercury thermometer as the gold standard. Pushing the thermometer probe by hand into the manatees mouth was performed with minimal effort. Proper placement was ensured by feeling for the most distal molar. Occasionally an animal would m ove their head slowly up and away from the direction of the probe. This behavior occurred wi th a few animals, but was never enough to requir e assistance in animal restraint, and all animals quickly relaxed following probe placement. The DS thermometers soft wire probe tip was chew ed open by one animal, exposing the underlying wires. The RS thermometers hard plastic probe tip was more resilient and did not break. Both probe types were relatively easy to maneuver by hand, as the loose skin around the manatees mouth provided adequate room along the jaw line. While we did not rigidly test for differences in temperature at the front of the mouth versus the back of the mouth, temperatures measured toward the back of the mouth were observed to be up to 0.5 C warmer than when the thermometer probe was displaced several centimete rs away, toward the front of the mouth. Heart Rate Monitoring and ECG Validation The stethoscope is traditionally the simplest m ethod to monitor HR. However, monitoring HR in exotic species has been accomplished with a variety of other instruments. Trained dolphins can be ECG monitored using a 2 lead set up. Suction cup electrodes can be placed along the ventral midline and the right scapula; or cros s chest electrodes can be held in place with a nylon strap (Noren et al 2004, Williams et al 1999). In elephants, a pulse oximeter clipped to the ear has proven effective (Osofsky 1997). Wire less wrist-watch style monitors have been

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26 used to monitor HR of deer (Montan et al. 2002). For the manatee, suction cup electrodes do not properly adhere to their irregul ar skin surface. Ventral placement of cross chest electrodes or small wireless monitors are a challenge to place on large manatees, requiring a team of handlers to properly position an animal, and risking the crus hing of equipment. A viable site for pulse oximetry has not yet been identified for the manatee. Monitoring HR via auscultation was the riskiest procedure as it placed the examiners head near the body of the manatee in a sometimes semi-prone position. Injury was prevented by sampling HR only when the manatee was fully secured, and not during an invasive sampling procedure such as venipuncture or tissue sampling. Manatees showed little to no irritation to repeated stethoscope placement. The only evasiv e behavior observed was a short slow turning of the body away from the direction of th e stethoscope by an individual. Failure of HR measurement vi a auscultation occurred. When the stethoscope bell was submerged in water, the gasket around the diap hragm of the waterproof stethoscope did not eliminate water flooding the bell. Position of the bell was important. Some animals required direct bell placement on the ventral midline, while placement proximal to the axilla was sufficient for others. Adequate pressure on the bell as well as placement of the bell was necessary for accurate auscultation. In this st udy weak sounding heart beats were often a result of poor bell placement rather than a sign of cardiac disease. Additionally, some individuals appear to be unable to auscult th e low frequency and/or decibel le vel of the manatee heart beat. Over the duration of this study, repeated pl acement of the stethoscope underneath manatees caused the researchers knuckles to be r ubbed raw, reddened, or slightly scarred. The performance of the ECG on manatees in the field, with lead set up as described by Siegal-Willott et al (2006) was generally accurate and re liable, with no permanent malfunctions

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27 or repairs needed. Manatees tolerated ECG monitoring quite we ll. Cleaning of the sites for electrode pad placement elicited no reaction. The occasional outli ers in ECG HR measurements were likely due to salt and sand contacting the electrodes and th ereby affecting electrode pad contact to the skin, as well as cont act of electrode pads to their lead wires. This can be corrected by cleaning, applying ultrasound ge l, and using tissue glue. Wh en proper lead connection is attained, HR by ECG and stethoscope are statistically and clinically similar. However, there are key issues which would favor one versus the othe r. A stethoscope should be used in conjunction with the ECG to ensure proper function. Once it has been determined through stethoscope that the ECG is measuring correctly, the ECG can effectively be used to continue to monitor HR. The benefits of ECG are its ab ility to provide a HR during transport by boat or car, where engine noise and vibration inhibit ausculta tion. Collected ECG tracings can serve as documentation of monitoring and enhance knowledge of heart function. The ECG has been used during transport of sick or injured manatees su ccessfully, allowing for HR to be monitored under noisy conditions, while limiting the mani pulation of the injured animal. ECG failure did occur approximately 15% of the time, generally due to water or sand contamination of the lead clips or electrodes. The beach environment with sand and salt spray was more challenging than workup sites on a boa t or paved ground. Warmer weather may allow for the adhesive electrode pads to better adhere to the manatees skin. Tissue glue on the electrode pads showed no peeling of the manatee skin when electrode pads were removed. Extra lead clips were kept on hand in th e field to readily replace lead c lips that were affected by excess water or debris. In general, the lead connection points tolerated the occasional fine mist of water spray and debris. Crossing of lead wires di d not seem to affect tracing printouts.

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28 There are some additional points that need a ttention during use of the ECG. Coordination of lead setup was necessary during processing an d sampling, as leads would occasionally detach due to manipulation of the manatee for variou s procedures. Shading of the ECG unit is recommended, as direct sunlight can wash out the ECG display. Paper printouts needed to be carefully prepared and stored in plastic bags to prevent them from becoming degraded by moisture and excessive handling. Analyses of pa per printouts needed to be performed manually. It is recommended that the researcher have a mi nimum of 30 cm in front of them for setting up the particular unit tested. The ECG can be exte nded 3-4 feet away from the animal. The ECG should be checked annually with an ECG simulator device to ensure proper electronic function. Future Considerations: Oral Temperature and Heart Rate Monitoring Oral tem perature and heart rate monitoring in manatees can effectivel y be performed with a basic digital thermometer and ECG in conjunction with a stethoscope. The ECG can be used while on a powered boat or in a moving transpor t truck, where noise and vibration can make it difficult to auscult via stethoscope The ECG should be used as a method to alert the researcher if HR falls out of a normal range. In such a case, immediate assessm ent should follow with a stethoscope as the gold sta ndard to confirm ECG measurements. A wireless thermometer transceiver unit with a hand held display console could improve versatility. A wireless ECG lead transceiver unit with a computer la p top display and storage is recomm ended for future field use.

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29 Table 2-1. Results of 0C water bath measurements by Radioshack (RS) and Digisense thermocouple (DS) thermometers Thermometer Mean Temperature ( SD); n=10 Range RS 63-1009A -0.2 ( 0.0) ----* RS 63-1035 (1) 0.5 ( <0.1) 0.4-0.5 RS 63-1035 (2) 0.2 ( <0.1) 0.2-0.3 RS 63-1035 (3) 0.2 ( 0.0) ----* RS 63-1035 (4) 0.5 ( 0.0) ----* DS (probe 1) 0.2 ( 0.1) 0.1-0.2 DS (probe 2) 0.1 ( 0.1) 0.0-0.3 The useful range for both thermometer types was 0 to 40C. RS and DS thermometers had a manufacturers specified accuracy of C and .4C respectively.* No range was determined as all temperature displays were identical. Table 2-2. Results of 15C wa ter bath measurements by Radioshack (RS) and Digisense thermocouple (DS) thermometers Thermometer Mean Temperature ( SD); n=10Range RS 63-1009A 14.3 ( 0.4) 13.4-14.5** RS 63-1035 (1) 15.0 ( 0.2) 14.5-15.2 RS 63-1035 (2) 14.7 ( 0.1) 14.5-14.9 RS 63-1035 (3) 14.7 ( 0.3) 14.0-14.9 RS 63-1035 (4) 15.1 ( 0.1) 14.9-15.3 DS (probe 1) 14.7 ( 0.9) 12.2-15.1** DS (probe 2) 14.8 ( 0.4) 13.6-15.0** The useful range for both thermometer types was 0 to 40C. RS and DS thermometers had a manufacturers specified accuracy of C and .4C respectively. ** Highlights values that were obtained outside of the thermometers respective accuracy. Table 2-3. Results of 30C wa ter bath measurements by Radioshack (RS) and Digisense thermocouple (DS) thermometers Thermometer Mean Temperature ( SD); n=10 Range RS 63-1009A 29.5 ( 0.2) 29.3-29.9 RS 63-1035 (1) 30.3 ( 0.2) 30.0-30.6 RS 63-1035 (2) 29.8 ( 0.2) 29.5-30.2 RS 63-1035 (3) 29.8 ( 0.2) 29.5-30.2 RS 63-1035 (4) 30.2 ( 0.2) 30.0-30.6 JTEK (probe 1) 30.0 ( 0.2) 29.7-30.3 JTEK (probe 2) 30.0 ( 0.2) 29.7-30.4 The useful range for both thermometer types was 0 to 40C. RS and DS thermometers had a manufacturers specified accuracy of C and .4C respectively.

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30 Table 2-4. Results of 45C wa ter bath measurements by Radioshack (RS) and Digisense thermocouple (DS) thermometers Thermometer Mean Temperature ( SD); n=10Range RS 63-1009A 44.0 ( 0.1) 43.8-44.1 RS 63-1035 (1)44.6 ( 0.2) 44.4-44.9 RS 63-1035 (2)44.1 ( 0.1) 44.0-44.2 RS 63-1035 (3)44.1 ( 0.1) 43.9-44.2 RS 63-1035 (4)44.6 ( 0.1) 44.4-44.8 DS (probe 1) 43.3 ( 3.5) 33.5-44.6** DS (probe 2) 44.3 ( 0.1) 44.2-44.7** The useful range for both thermometer types was 0 to 40C. RS and DS thermometers had a manufacturers specified accuracy of C and .4C respectively. ** Highlights values that were obtained outside of the thermometers respective accuracy. Table 2-5. Results of 60C wa ter bath measurements by Radioshack (RS) and Digisense thermocouple (DS) thermometers Thermometer Mean Temperature ( SD); n=10Range RS 63-1009A 59.05 ( 0.5) 58.4-59.9** RS 63-1035 (1) 59.74 ( 0.3) 59.4-60.2 RS 63-1035 (2) 59.16 ( 0.2) 58.8-59.5 RS 63-1035 (3) 59.27 ( 0.3) 58.9-59.9 RS 63-1035 (4) 59.76 ( .0.2) 59.4-60.0 DS (probe 1) 59.60 ( 0.4) 59.3-60.6** DS (probe 2) 59.42 ( 0.3) 59.1-60.0** The useful range for both thermometer types was 0 to 40C. RS and DS thermometers had a manufacturers specified accuracy of C and .4C respectively. ** Highlights values that were obtained outside of the thermometers respective accuracy. Table 2-6. Results of 75C wa ter bath measurements by Digisense thermocouple (DS) thermometer Thermometer Mean Temperature ( SD); n=10 Range DS (probe 1) 74.5 ( 0.8) 73.4-75.8** DS (probe 2) 74.7 ( 0.9) 73.3-76.1** The useful range for the Digisense thermocouple (DS) thermometer was 0 to 40C. The DS thermometer had a manufacturers specified ac curacy of .4C respectively. ** Highlights values that were obtained outside of the thermometers respective accuracy.

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31Table 2-7. Evaluation of ECG pe rformance on 25 manatees Animal ID Straight len g th ( cm ) Weight ( k g) Sex Date Successful r eadin g Printer j a m Lead m alfunction Comments TTB119 251 361 F 05 Dec 2005 2 1 Not reading, loose connection with plug, replaced electrode clip. TTB120 259 345 M 05 Dec 2005 2 TTB122 276 474 F 06 Dec 2005 5 1 TTB123 292 551 F 06 Dec 2005 3 2 CTB051 231 470 M 05 Jan 2006 6 TTB125 277 401 M 04 Jan 2006 5 1 TTB101 290 460 M 05 Jan 2006 1 1 Tracings 2 and 3 not showing for augmented print. TTB128 296 506 M 04 Jan 2006 11 TTB132 302 816907* F 05 Jan 2006 2 3 1 Electrode clips replaced TTB127 313 540 M 04 Jan 2006 1 TTB006 316 540 F 05 Jan 2006 5 1 ECG searching for signal. Added gel to electrode terminals. BZ05F78 191 132 F 14 Nov 2005 2 BZ05F92 204 166 F 17 Nov 2005 1 BZ03F28 210 190 F 15 Nov 2005 2 n=11 Florida manatees (TTB and CTB prefix IDs) and n=14 Antillean manatees (BZ prefix IDs). A range of weight is given because lifting chains broke while lif ting animal in stretcher for weight.

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32 Table 2-7. Continued Animal ID Straight-line length (cm) Weight (kg) Sex Date Successful Reading Printer Jam Lead Malfunction Comments BZ05F89 215 182 F 16 Nov 2005 7 BZ98M05 235 289 M 14 Nov 2005 1 BZ01M15 240 228 M 16 Nov 2005 2 BZ05M85 245 323 M 12 Nov 2005 5 1 3 Checked leads. Salt in electrode clips-replaced BZ05F86 248 268 F 13 Nov 2005 2 1 ECG searching for signal, clips were wet, had to replace BZ05F91 251 279 F 17 Nov 2005 7 1 Electrode clips wet-replaced BZ97F03 252 309 F 14 Nov 2005 2 1 ECG searching for signal. Electrode clip came off-replaced BZ05M93 261 327 M 18 Nov 2005 2 BZ05M88 259 323 M 16 Nov 2005 2 1 Electrode clip came off-replaced BZ05M90 284 409 M 16 Nov 2005 6 BZ05M87 291 424 M 15 Nov 2005 13 TOTAL 97 8 11 Percentage 83.6 6.9 9.5

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33 CHAPTER 3 ORAL TEMPERATURE, HEART RATE, AN D RESPIRATION RATE OF MANATEES (T richechus manatus latirostris, T. m. manatus ) EXPOSE D TO CAPTURE AND HANDLING IN THE FIELD Abstract Many m anatee research studies require the cap ture and handling of animals to collect data. Understanding how manatee oral temperature (OT), heart ra te (HR), and respiration rate (RR) can change during a capture event is important for resear chers who want to ensure an animals well-being. To determine the effect s of capture on healthy, awake, juvenile/adult manatee vital signs: a total of 38 Florida manatees (Trichechus manatus latirostris ) and 48 Antillean manatees ( T. m. manatus ) were continuously monitored for OT, HR, and RR during field capture events. Creatin e kinase (CK), potassium (K+), serum amyloid A (SAA), and lactate values were examined for each animal to assess possible systemic inflammation and muscular trauma. Manatee OT, HR, and RR all changed ov er a 50 minute period, with Antillean manatees having higher parameters than Florida manatees. Mean (SD) OT of Florida and Antillean manatees increased during capture from 32.6 (.8) C and 34.6 (.9) C; to 34.8 (.5) C and 35.2 (.7) C respectively. Animals were removed from water temperatures of 25.9 (.2) C in Florida; and 28.9 (.5) C in Belize and Puerto Rico. There wa s no statistically significant difference of OT between Antillean and Florida manatees after 40 minutes ( p=0.4186). Mean (SD) HR of Florida and Antillean manatees d ecreased from 66 (.7) beats per minute (bpm) and 75 (.7) bpm to 60 (.9) bpm and 61 (.5) bpm, respectively. There was no statistically significant difference of HR between Florid a and Antillean manatees after 25 minutes ( p=0.0739). Mean (SD) RR of Florida and Anti llean manatees decreased from 6 (.9) breaths/5min and 9 (.5) breaths/5min to 4 (.0) breaths/5min and 5 (.3) breaths/5min, respectively. There was no significant difference in RR after 50 minutes be tween Florida and

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34 Antillean manatees ( p=0.0014). Higher respiratory rate ove r time was associated with higher lactate values. Antillean manatees had higher ove rall lactate values than Florida manatees ( p<0.001). Monitoring of manatee OT, HR, and RR in the field is recommended as standard protocol for researchers, to be tter assess the condition of an animal. Introduction The Florida m anatee, Trichechus manatus latirostris, has a minimum population size calculated to be 3,300 animals (Haubold et al. 2006). It is classified as an endangered species in the State of Florida, protected by the U.S. Enda ngered Species Act, and by the Marine Mammal Protection Act, listed in the Convention on International Trade of Enda ngered Species of Wild Flora and Fauna under Appendix I, and defined as vulnerable by the In ternational Union for Conservation of Nature and Natural Resources (U.S. Fish and Wildlife Service 2004, Convention on International Trade of Endangered Speci es of Wild Flora and Fauna 2004, Haubold et al 2006, International Union for C onservation of Nature and Natural Resources 2007). Since the early 1970s multiple federal, state, private, and non-profit organizations have been working together to protec t and conserve the species from anthropogenic and natural threats to its population. As part of the conservation e ffort, healthy free-ranging manatees are routinely captured for population studies and research purposes. Between the years 1975 and 1983, OShea et al. (1985) reviewed net captures of 92 manat ees in Florida. Based on blood chemistry parameters including aminotransferase activity, creatine kinase, potassium, and lactate, they determined that both healthy and distressed manat ees were generally not susceptible to severe capture stress. Since the late 1970s, only six manatees have died in over 1000 capture events (U.S. Geological Survey, Florida Fish and Wild life Conservation Commission, Wildlife Trust, unpublished data). Upon necropsy,of the carcasses symptoms of capture myopathy as defined by Spraker (1993) were not found.

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35 While capture related deaths ar e low, field researchers encounter animals during research captures and rescues with symptoms such as br adycardia, apnea, hyperthermia, and hypothermia. Recognizing these physiological abnormalities is important to biologists and veterinarians who want to improve manatee triage and husbandry in the field. Understa nding oral temperature (OT), heart rate (HR), respiration rate (RR) and blood chemistry parameters of manatees under various field circumstances may aid with the interpretation of physiologic and pathologic findings. The purpose of this research was to determine normal ranges and dynamics of manatee OT, HR, and RR over time during field captures and determine their relationships with creatine kinase (CK), potassium (K+), serum amyloid A (SAA), and lactate. Materials and Methods During the years 2004-2006, free ranging m ana tees were captured at the following locations: Florida : Apollo Beach, in Tampa Bay, n ear the TECO Big Bend power plant (27.01N, 82.87W); Port of the Islands residential and marina basin, in Naples, Faka Union Canal (25.74 N, 81.03W) ; Everglades National Park, Whitewater and Coot bays (25.77N, 80.37W). Belize : Inshore waterway of Southern Lagoon (17.21N, 88.78W); and offshore isles of the Drowned Cays (17.25N, 8823.40W). Puerto Rico : Boqueron, Rincn Canal (18 2.32N, 67.15W); Guayanilla, Bahia de Guayanilla (17.49N, 66.64W); Ceiba, near-shore waters off former Naval Station Roosevelt Roads (18.40N, 65.71W). Captures were performed under United States Fish and Wildlife Service research permits MA773494 (Florida Fish and Wildlife Conservation Commission), M791721/4 (U.S. Geological Survey), MA791721/4 (U.S. Geological Survey), and the Belizean Department of Forestry

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36 permit CD/60/3/05(36) (Wildlife Trust). Manatees in this study were concurrently monitored for OT, HR, and RR, sampled for research purposes a nd given complete health assessments. A total of 86 apparently healthy, free-ranging, juvenile and adult manate es were monitored for OT, HR, and RR in this study, 38 were Florida manatees and 48 were Antillean manatees (Table 3-1). Capture of manatees consisted of efforts from many people through the use of a capture boat, support boats, and aerial observation. The capture boat was a motorized, modified mullet skiff with a removable transom, a raised control cons ole for enhanced viewing, and a large seine net with surface floats and a lead weighted bottom. One to three small motor boats provided on the water safety support, assistance in locating animals, and additiona l animal handlers once the net was set on the manatee. A capture crew consisted of a minimum of ten people. To help locate manatees from the air, an observer in a small plane or helicopter was used when possible. Manatees were selected visually based on indi vidual size and number of grouped individuals. Preference was given to larger animals and effort s were made to avoid harassing or capture of mother-calf pairs. Once located, the target ma natee was encircled by the capture boat, in a number 30 or 56 braided seine twine net approximately 122-183 meters long, approximately 8 meters deep, with an approximately 10-17 cm stretch knotted nylon mesh. The net and manatee were then pulled into the boat. Slight modifi cations to the basic capture method occurred in Florida and Belize. In Florida, manatees were usually captur ed using land based net sets, simultaneously entrapping one to four animals, and in one instance up to 12 manatees along a shoreline. In Belize, one to three manatees were typically caught in larg e open water net sets at one time. The manatees were corralled into a sm aller net with a bag end, guided to the stern of the boat, and pulled aboard. If ma natees needed to be moved to a work-up site, they were placed in a stretcher and carried by handlers. Capture times varied.

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37 Monitoring of OT, HR, and RR began immediately upon safely securing the manatee on the deck of the capture boat or on land. OT HR, and RR were monitored continuously during the holding period until the manatee was released. A Radioshack (RS) thermometer model 63-1009A and RS thermometer model 631035 were used to measure oral temperature. Th ermometers were lab tested to ensure accuracy prior to field deployment (CHAPTER 2). OT pr obes were placed once the manatee was secured on land or on a boat deck. The soft wire te mperature probe was placed by hand, along the jaw line, buccally past the rear mandibular molars (C HAPTER 4). OT values were recorded every 5 minutes simultaneously with HR and RR. The te mperature probes were removed just prior to release. A Sonocardia waterproof stethoscope (Magnafortis Seattle, WA) was used to auscult the heart. The stethoscope bell was inserted und er the manatees axilla and placed ventrally, close to midline. The number of heart beats, de fined as atrial and ventri cular contractions, were counted for 15 seconds and multiplied by four to determine beats per minute (bpm). HR was recorded every 5 minutes, in conjunction with OT a nd continuous RR monitoring. A Mac 500 electrocardiogram (ECG ) (GE Healthcare, Milwaukee, WI ) was used to assess heart function and HR. Skin with dirt or slime was scraped, wiped with an alcohol pad, and dried extensively with an absorbent sponge, heavy duty paper towels, or beach towel, to ensure proper adherence of sticky electrode pads Ultra sound gel was used at the connection point of the lead clip and the electrode pad to improve electrical conductivity. A small amount of tissue glue was applied to the cleaned site. ECG HR was compared to stethoscope to ensure its accuracy. Printed ECG tracings were examined to ensure validity of the units displayed HR and to assess heart function (Siegal-Willott et al 2006). ECG tracings that showed artifacts such

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38 as distorted baselines, or unclear PQRS complexes were consid ered as evidence for an inaccurate HR reading. Data from inaccurate tracings were not used and these were considered equipment failures. Monitoring of respiration began when a mana tee was secured on a boat or land, and ended upon release back into the water. Time and qua lity of breaths were recorded. Evidence of respiration needed to be seen, heard, or felt. Opening of the nares alone did not constitute a breath. Visible respiratory ex change was determined by thor acic excursion. Sounds of respiratory exchange were also considered as evidence of breath ing. A hand was placed in front of a manatees nares to feel for an exhalation or inhalation. Respiratory abnormalities such as air leaking out of closed nares we re described and recorded. Lu ngs were ausculted dorsally to assess respiratory health. The following defined terms were commonly used to help describe observed quality of breaths. Shallow breath: A relatively short respiratory exch ange time is observed, heard, or felt. There is little rising or lowering of the manatees thoracic region. Deep breath : A relatively long respiratory exchange time is observed, heard, or felt. The rising and lowering of the mana tees thoracic region is qui te obvious and great. Stuttering breath : Obvious, short, rapid cessations of inhalation occur during inhalation. No exhalation during the ce ssations of inhalation. Blood Sampling and Biochemical Analysis Blood was sam pled from the brachial vascular bundle, located betw een the radius and ulna (Medway et al .1982; Bossart et al 2001). The medial or lateral, mid-pectoral region of the manatee was manually restrained and sterilely prepared by alternating Betadine, a 10% povidone iodine solution (Purdue Freder ick Company, Norwalk, CT), and 70% isopropyl alchohol three times in concentric circles radiating outwards. Vacutainer equipment was used with a 21 gauge, 1.5 in needle with a syringe or extension set. Blood samples were generally collected within 15

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39 to 45 minutes of capture. Total time of blood collection varied from 5 minutes to 30 minutes, depending on the flow of blood into the tubes, th e additional number of tubes being filled for other studies, and the activity of the animal. Ethyoenediamine tetra-ac etic acid (EDTA) tubes and lithium heparin tubes were gently inverted for 2 minutes after collection. Tubes with anticoagulants were placed in a c ooler, on top of a small foam pad on ice, as soon as possible after collection. Plasma and serum were separated within 1 hour of collection. Samples were transported on ice at 4 to 10C until laboratory analysis. Belize serum and plasma samples were stored at -20C prior to analysis. Complete blood count, biochemical analysis and protein gel el ectrophoresis were performed at the University of Florida-College of Veterinary Medici ne Clinical Pathology Laboratory. Creatine kinase, potassium (K+), serum amyloid A (SAA), and lactate values were determined as described by Harr et al (2006) and Harvey et al (2007). Statistical Analyses Statistical analyses were conducted us ing Microsoft Office Excel 2003 for W indows (Microsoft Corp., Redmond, WA), and Sigma Stat for Windows, Version 3.1 (Systat Software Inc., Point Richmond, CA). T-tests were conducted at a significance level (Type I error rate) of = 0.05 whereby, the null hypothesis was define d as the two groups lacking significant difference. Correlation and regression analyses were conducted whereby an r2 = 0.5 or greater was considered a significant relationship. Only complete OT, HR, and RR data were used in the statistical analysis. Complete OT, HR, and RR was defined as: monitoring where no mo re than 10 minutes of continuous time, or two consecutive 5 minute time intervals, were missed during a 50 minute monitoring session. While some manatees were monitored for over 50 minutes, the sample size supported statistical analysis up to 50 minutes. Antillean manatees captured from Puerto Rico and Belize were

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40 grouped together for statistical comparison with Fl orida manatees. A repeated measures linear mixed model (Proc Mixed; SAS Institute, Car y, NC) was used to determine if there was a significant difference between the OT, HR, and RR of manatees based on the fixed effects of time, location, sex, and size class. Type 3 sums of squares and differences of the least square means were calculated for significant fixed effects. Correlation analyses of HR versus RR, for Florida and Antillean manatees were conducted. Unpaired t-tests were conducted between respective Florida and Antillean: CK activity, K+, SAA, and lactate values. Based on normal Florida reference intervals (Harvey et al 2007), manatees with CK activity > 482 U/L, K+ > 6 mEq/L, SAA> 70 ug/mL, and l actate values >25 mmol/L were examined for abnormal OT, HR, and RR. Regr ession analyses were performed between OT, HR, and RR versus blood chemistry. Unpaired ttests were used to compare the means of OT, HR, and RR values in groups of animals with normal and abnormal biochemical values. Average HR and RR over 20 minutes and 40 minutes were grouped into those with six breaths or higher versus those with five breaths or fewer and at 70 bpm or higher versus 69 bpm or less, respectively. An unpaired t-test was then used to compar e the mean lactate concentration within these groups. Results Capture Clim ate conditions for captures were distin ctly different for Florida and Antillean manatees. In Florida, mean air temperature was 23.8 (.0) C, and ranged from 14.7C to 31.9C; mean water temperature was 25.9 (.2) C, and ranged from 21.5C to 30.9C. In Belize and Puerto Rico combine d, mean air temperature was 30.7 (.9) C, and ranged from

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41 24.2C to 40.8C; mean water temperature was 28.9 (.5) C, and ranged from 25.1C to 31.5C. Capture times averaged 43 minutes from the time of net set to when the manatee was secured for examination, for Florida and Belize. Th is was due to the logistical complexity that occurred when multiple manatees were netted. In Puerto Rico, where single individuals were captured at a time, animals were secured within 5 minutes of being netted. In TECO Tampa Bay, on 14 December 2007, 12 manatees were netted at one time in a land set. Six individuals from this group were monitored for this study. For approximately 4.5 hours, several individuals were maintained in water that was just deep e nough to be fully submerged, prior to being moved for examination. Similarly, in Belize several an imals swam around in the large net for a little over 2.5 hours before being secured for examination (Table 3-7). Oral Temperature No significant difference in OT over time was found between adu lt and juvenile size classes, or between sexes. Initial OT of the Florida manatees averaged 32.6 (.8) C, with a range of 29.5C to 35.1C. The final 50 minute interval OT of the Florida manatees averaged 34.8 (.5) C, with a range of 30.5C to 35.9C. Initial OT of the Antillean manatees averaged 34.6 (.9) C, with a range of 32.6C to 36.0 C. The final 50 minute interval OT of the Antillean manatees averaged 35.2 (.7) C, with a range of 34.0C to 36.1C. Initially, OTs between the two subspecies we re significantly different (p<0.0001). By the 40 minute time interval, there was no statistically significant difference in OTs between Florida and Antillean manatees, with average OTs of 34.2 (.6) C and 35.2 (.6) C, respectively (p=0.4186) (Figure 3-2, Table 3-3). A total range of 29.5 C to 36.2 C was observed from Antillean and Florida manatees.

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42 Heart Rate No significant difference in HR over tim e was found between adu lt and juvenile size classes, or between sexes. In itial HR of the Florida manatees averaged 66 (.7) bpm, with a range of 49 bpm to 80 bpm. The final 50 minute in terval HR of the Florida manatees averaged 60 (.9) bpm, with a range of 45 bpm to 68 bpm. Initial HR of the Antill ean manatees averaged 75 (.7) bpm, with a range of 64 bpm to 88 bpm. The final 50 minute interval HR of the Antillean manatees averaged 61 (0.5) bpm, with a range of 48 bpm to 84 bpm. Initially, there was a significant difference between the HRs of the two subspecies (p=0.0058). However, by 25 minutes there was no statistically significant di fference between Florida and Antillean manatees, with average HRs of 60 (.6) bpm and 63 (.1) bpm, respectively ( p= 0.0739) (Figure 3-3, Table 3-4). A total range of 32 bpm to 88 bpm was observed from Antillean and Florida manatees in the field. Respiration Rate No significant difference in RR over time was found between adu lt and juvenile size classes, or between sexes. Initial RR of the Florida manat ees averaged 6 (.9) breaths/5 minutes, with a range of 1 breath/ 5 minutes to 13 breaths/5 minutes The final 50 minute interval RR of the Florida manatees averaged 4 (.0) br eaths/5 minutes, with a range of 0 breaths/5 minutes to 8 breaths/5 minutes. Initial RR of the Antillean manatees averaged 9 (.5) breaths/5 minutes, with a range of 3 breaths/5 minutes to 17 breaths/5 minutes. The final 50 minute interval RR of the Antillean manatees averaged 5 (.3) breaths/5 minutes, with a range of 2 breaths/5 minutes to 12 breaths/5 minutes. Initially, respiration rates between the two subspecies were significantly different ( p<0.0001). Over time the respirat ion rates of Antillean and Florida manatees became progressively lower. Howe ver, by 50 minutes there was no statistically significant difference between Florida and Antill ean manatees with average RRs of 4 (.0)

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43 breaths/5 minutes and 5 (.3) br eaths/5 minutes, respectively (p=0.0943) (Figure 3-4, Table 35). A total range of 0 breaths/5 minutes to 17 breaths/5 minutes was observed from Antillean and Florida manatees. A positive correlation existed between heart rate and respiration rate in manatees. Florida manatees had an r2=0.732 and Antillean manatees had an r2=0.8598 (Figure 3-5). Blood Chemistry: Creatine Kinase, Po tassiu m, Serum Amyloid A, and Lactate There was no statistically significant differe nce between Florida and Antillean measured CK activity. Mean CK activity of 38 Florida animals was 193 () U/L with a range of 51799 U/L. Mean CK activity of 48 Antillean manatees was 195 () U/L with a range of 38 U/L to 3522 U/L. Within theses sampled gr oups, two Florida manatees and one Antillean manatee had abnormal values with a combined range of 572 U/L to 3522 U/L. There was no statistically significant differe nce between Florida and Antillean potassium values. Mean K+ of 38 Florida manatees was 5.1 (.6) mEq/L with a range of 3.9 mEq/L to 6.4 mEq/L. Mean K+ of Antillean manatees was 5.3 (.5) mEq/L with a range of 4.1 mEq/L to 7 mEq/L. Within these sampled groups three Florida manatees and three Antillean manatees had abnormal values with a combined range of 6.1 mEq/L to 7 mEq/L. There was no statistically significant diffe rence between Florida and Antillean SAA values. Mean SAA for Florida manatees wa s 97 ( 235) ug/ul with a range of 10 to 1200.ug/mL). Mean serum amyloid A for Antillean manatees was 25 (2) ug/mL with a range of 10 ug/mL to 190 ug/mL. Within these two sa mpled groups nine Florida manatees and four Antillean manatees had abnormal values with a combined range of 73 ug/mL to >1200 ug/mL. A statistically significant difference existed between mean lactate values of Florida (n=37) and Antillean (n=44) manatees, of 13.7 ( 6.7) mmol/L and 20.6 ( 7.8) mmol/L, respectively (p<0.001). Ranges of lactate values were 0.5 mmol/L to 31 mmol/L and 5

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44 mmol/Lto 37 mmol/L for Florida and Antillean ma natees, respectively. Fourteen manatees presented lactate levels great er than 25 mmol/L, with a total range of 26.5 mmol/L to 32 mmol/L). Twelve of the 14 manatees were Antillean. Respiration Rate and Lactate A student' s t-test revealed a statistically significant difference between lactate values of all manatees based on mean RR at 20 minutes, of 5 breaths (n=24) versus 6 breaths (n=57) ( p=0.020). Whereby, mean lactate (SD) was 14.3 ( 7.6) mmol/L and 18.8 ( 7.9) mmol/L, respectively. A statis tically significant difference was also observed when conducting a students t-test comparing lactate values of a ll manatees based on mean respiration rates in 40 minutes, of 5 breaths (n=32) versus 6 breaths (n=49) ( p=0 .018). Whereby, mean lactate (SD) was 14.9 ( 7.8) and mmol/L 19.1 ( 7.8) mmol/L, respectively. Sample size was not sufficient enough to compare lactate values based on respiration rates within the two subspecies. No significant correlation wa s observed between lactate vers us HR and RR. However, a female Antillean manatee, BZ97F03, captured on 3 separate occasions showed a positive correlation between initial HR, initial RR, and lactate values (Table 3-6). Other Significant Findings A 231 cm long male (CTB051), captured in Tampa Bay on 05 January 2006, demonstrated a pronounced and persistent bradycar dia, with irregular rhythm, an instantaneous HR of 42 bpm, and a mean of 48 bpm on the ECG. Heart rate was take n by stethoscope seven times during a 5 minute period and ranged betw een 28 bpm and 48 bpm. RR during this time was 4 breaths/5 minutes. Thirty minutes later, the HR stabilized at 60 bpm, RR was 6 breaths/ 5 minutes. Oral temperature ranged between 34.1 C to 34.6 C. The SAA was 83 ug/mL. A 289 cm long female (TNP029), captured in Port of the Islands on 18 April 2004 was found dead 32 days later from a chronic infection due to a watercraft impact related injury. No

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45 obvious signs of illness were apparent upon cap ture. However, the SAA value was >1200 ug/mL. At necropsy, a pyothorax was confirme d (Florida Fish and Wildlife Conservation Commission, unpublished data). The manatees OT was 34.7 C, HR ranged from 52 bpm to 60 bpm, and RR ranged from 2 breaths/5 mi nutes to 15 breaths/5 minutes. Discussion Capture The groupin g of Antillean manatees captured by corralling into a bag net in Belize, and simpler open water net sets in Puerto Rico, was supported by the lack of a significant differences between OT, HR, and RR of the two groups. C onsidering how distinctly different these two capture methods were, the results suggest that the differences in OT, HR, and RR between Florida and Antillean manatees may also be due to other capture related factors such as pursuit time by boat prior to net set, struggl e duration and intensity, or indivi dual degrees of excitability. While pursuing manatees for capture, focal follo w of individuals underwater was challenged by poor water visibility, and assessment of struggle duration and intensity was considered too subjective. Furthermore, the instances when th ere was a preference to pick more robust animals and avoid mother-calf pairs, did not create a samp ling bias as the resulting animals sampled were relatively well distributed in size and sex. Oral Temperature Oral temperature monitoring is an effective t echnique to ascertain thermal homeostasis in a manatee, but should not necessarily be cons idered an exceptionally sensitive method of measuring body heat build up. Oral temperature ranges in this study did not exceed the reported normal OT range of 35.5C to 36.0C (Murphy 2003). The results fr om this study suggest that captured manatees are effectively managing near or within the reported core body temperature of 35.6C to 36.4C (Gallivan et al 1983; Irvine 1983), and/or that OT may be physiologically

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46 limited in terms of the maximum level of heat that is distributed to the buccal area in exercised, healthy individuals. Measurement of fluke temperature in conjunction with oral temperature may prove useful to better assess thermal insult in field captured manatees. When forced to dissipate body heat, manatees are able to increase arte rial blood flow to the skin of the fluke, via regulation of blood flow through deep caudal veins located collateral to the caudal vascular bundle of the chevron canal (Rommel & Caplan 2003). Heart Rate Manatee mean HR following 50 minutes post capture monitoring returned to near the normal resting max HR of 60 bpm reported for captive manatees in the water and on land (Scholander & Irving 1941, Murphy 2003). These results suggest that manatees are not reacting with a tachycardia in terms of a fright response, or a bradycardia in terms of a dive response. In this study, initial individual HR measurements were as high as 88 bpm. Previous captive manatee HR studies demonstr ated a HR of 70 bpm upon surfacing from a 5 bpm dive bradycardia, and 75 bpm while being monitored with an ECG in a drained pool (Tenney & Hanover 1958, Gallivan et al. 1986). The elevated initial HR of captured manatees is likely a physiological response in order to s upply the increased demand for oxygen and energy by active muscles. Additionally, increased blood flow to the fluke and other extremities in response to increased body heat, may also have contributed to an incr ease in HR. In general, this study did not see profound bradycar dic dive responses or fright responses as suggested by Butler (1982), and demonstrated by Gallivan et al (1986) on manatees in wate r. These results suggest that manatees may recognize when they are on land and in the water, and have different responses for the two different environments.

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47 Respiration Rate Initial RR of captured manatees was significantly higher than the norm al 2 breaths/5 minutes to 4 breaths/5 minut es (Scholander & Irving 1941, Murphy 2003, Bossart 2001). This studys RR range of 0 breaths/5 minutes to17 br eaths/5 minutes exceeded the RR observed by Walsh & Bossart (1999) of 3 br eaths/5 minutes to15 breaths/5 minutes, of manatees being assessed on land. It is likely th at the 10 minute to 16 minute periods of breath holding recorded in previous dive studies is si gnificantly reduced following intens e struggle in the field captured manatee (Scholander & Irving 1941, Gallivan et al 1986). Increased rhythmic respirations have b een observed in bottom resting manatees submerged for extended periods of time (Hartm an 1979, Parker 1922). It is likely that the respirations observed in captured manatees were stronger and deeper than those of the bottom resting manatee. The increase in minute ventilation is likely a hypercapnic response in the captured manatee, whereby high CO2 and not low O2 was the triggering factor (Gallivan & Best 1980). Hyperventilation in manatees rapidly re plenishes oxygen stores while eliminating carbon dioxide and is typical of sl ow breathing marine mammals (K ooyman 1973). In this study, RR was generally elevated above reported normal valu es. Also, many animals did not return to the reported normal RR during monitoring. The persis tent elevated RR overtime suggests that full recovery from struggle can take over 50 minutes. Captured mana tees released within an hour may still be in a recovery stage upon release. Difficulty in a breathing response followi ng a forced submergence of a 12 minute to15 minute duration was observed in a 330 kg individual (Scholander & Irving 1941). Following this extreme dive duration, respirations increased to two or three breaths per minute. Apnea and decreased respirations were occa sionally observed in captured manatees, but not in association with a bradycardia of 40 bpm. This observation however, does not preclude the possibility of a

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48 dive response in manatees. Manatees with an RR below 2 breaths/5 minutes were stimulated to breathe by pouring water over the back of the hea d. The active induction of respirations is an animal care technique used by field researchers to ensure that a captured manatee is adequately ventilating, and may have prevented a dive response from occurring during this study. Blood Chemistry: Creatine Kinase, Pota ssiu m, Serum Amyloid A, and Lactate Standard manatee hematological and bioc hemical references are relatively well established (Medway et al 1982, Walsh & Bossart 1999, Murphy 2003, Harvey et al 2007). Furthermore, research has been conducted on specific blood parameters to support a more diagnostic approach to manatee health in the field. The evaluation of CK, K+, SAA, and lactate are used to assess muscle damage and systemic inflammation associated with exertional or pathological distress. CK is an enzyme commonly found in the cytopl asm of muscle cells. Its primary function is to catalyze the formation of phosphocreatine, a source of energy for muscle activity. CK has a relatively short half life in ma ny mammals, but specific duration has not been determined in manatees. High levels of CK activity mainta ined overtime are generally associated with continual muscle damage from muscle diseas e and exertion. The reported normal range of manatee creatine kinase (CK) activity is 79 U/ L to 302 U/L (Walsh & Bossart 1999). Current reported normal mean CK values for two captiv e adult manatees are reported by Manire et al (2003) as 223.8 U/L and 233.8 U/L. CK in a free-ranging captured manatee (8247B) following severe struggle was observed as high as 1365 U/L sampled the same day, and 1381 U/L 3 days later while in captivity (OShea et al 1985). One hundred days later, the same individuals CK activity was 227 U/L. This re sult was common amongst other sim ilar individuals in the study. While it has been suggested that manatees are likely not susceptible to capture myopathy, such elevated levels of CK activity are indicative of muscle damage (Harvey et al. 2007). Chronically

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49 injured manatees may show mildly increased CK activities. For example, a rescued manatee with an embedded line entanglement to its pect oral flipper (SWF-TM-613B) showed a CK value of 520 U/L (Beusse et al unpublished data). Potassium is an electrolyte that helps to maintain cellular osmotic balance, and is mostly found in muscle cells. High levels of serum K+ can be associated with ac idemia, as intracellular K+ are exchanged for H+ in the blood to maintain cellular osmotic balance. The reported normal range of K+ is 4.2 mEq/L to 6.6 mEq/L (Walsh & Bossart 1999). White et al (1976) reports normal mean (SD) for Na+ as 143.8(.20) mEq/L and for K+ as 5.0 (.14) mEq/L. Similarly, Medway et al (1982) show respective values of 1 51 (.3) mEq/L and 5.2(.7) mEq/L. Harvey et al (2007) show similar results for both free ranging and captive manatees. Serum amyloid A is an acute phase response protein which increases during acute inflammation and infection. Harr et al (2006) established use of the acute phase response protein SAA as a general indicator of tissue damage in manatees. In their research, healthy manatees were determined to have a mean (SD) SAA of 22 () g/mL. In comparison, diseased manatees showed a mean SAA value of 266 () g/mL. Lactate is a biochemical byproduct of the anaer obic metabolism of glucose. Increased levels of lactate are positively associated with increased levels of muscular exertion, which can contribute to metabolic acidosis. In their manatee diving experiment, Scholander and Irving (1941) observed lactate levels si gnificantly increasing from 1.11 mm ol/L pre dive to a range of 11.1 mmol/L to 16.65 mmol/L post dive. Increases in lactate occurred a few minutes after the dive, but not during the dives. Scholander a nd Irving (1941) explain that such phenomena is indicative of peri-vascular constr iction of circulation within th e muscle tissue during the dive, isolating lactic acid build up from the general circulation. Inverse to blood O2, CO2 increased

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50 during dives and decreased duri ng dive recovery. Harvey et al (2007) found that mean lactate in normal free ranging manatees was 13 mmol/L, a nd 4 mmol/L in normal captive manatees. One should consider that the increased lactate c oncentration in free ranging manatees may be associated with struggle due to capture. In this study, the majority of captured mana tees presented normal blood chemistry values and were considered healthy animal s. Animals with abnormal CK, K+, and SAA did not have associated abnormal OT, HR, or RR. However, significant associations were made between lactate and RR. Respiration rate and lactate The underlying cause of m etabolic ac idosis is an over abundance of H+ protons resulting from glycolysis and ATP hydrolysis, due to anaerobic metabolism (Robergs & Roberts 1997). Clinically, metabolic acidosis has been traditi onally and indirectly asse ssed by the associated symptomatic increase in levels of lactate. Lact ate values from captured manatees show a range resembling that of mild, moderate, and extrem e levels of exertion for thoroughbred horses (Bayly et al .1983, Bayly et al 1987, Rose et al 1988). Harvey et al (2007) show that high lactate values in free-ranging Florida manatees are associated with low plasma CO2, which would be attributed to increased respiration rate to blow off HCO3 and maintain optimal blood pH (Robergs & Roberts 1997). Similarly, Scholander and Irving (1941) observed in manatee recovery from forced dives a return to predive blood O2 levels, and a decrease in blood CO2 with an associated increase in lactic acid. RR levels show potential to serve as predictors of lactate levels in captured manatees. BZ97F03, captured on three separate occasions, s howed lactate levels of 27.5 U/L, 20 U/L, and 9.7 U/L. Concurrently, initial HR and RR values were 92 bpm and 16 breaths/5 minutes; 84 bpm and 8 breaths/5 minutes; 60 bpm a nd 3 breaths/5 minutes respectivel y. Individuals with elevated

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51 RR levels that do not decrease over a 50 minute tim e period should be clinically assessed by a veterinarian. Such individuals are not properly adjusting and returning them to the water is recommended. Florida and Antillean Manatees: Differen ces in Oral Te mperature; Heart Rate, Respiration Rate, and Lactate Oral Temperature The initial o ral temperature differences be tween the Florida and Antillean manatees may in part be due to differences in subcutan eous fat thickness, body condition, and water temperature. Gallivan et al (1983) notes an individual mana tees core temperature at 33.0C and falling in a water temperature of 23.0C. At the same length and after gaining 26 kg in body weight, the same animal maintained a core temperature of 34.0 C in a water temperature of 20.0C. In respect to these findings, Antillean manatees may be leaner than Florida manatees as part of an adaptation to their warmer environm ent, and possibly have a higher minimal water temperature limit than the 20.0 C reported for Florida manatees (Irvine 1983, Bossart 2001). However, there is currently not a large enough data set regarding length, weight, and subcutaneous fat measurements of Antillean manat ees to compare them with Florida manatees. Heart Rate, Respiration Rate, and Lactate Lower HR, RR, and lactate in F lorida manatees compared to Antillean manatees may be attributed to better endurance conditioning in Flor ida animals. Florida manatees can have long distance seasonal migrations generally between <50km to 830 km, with a unique individual traveling greater than 2,300 km (Deutsch et al 2003). Populations in Puerto Rico and Belize generally remain within a 50 km home range (OShea & Salisbury 1991, Lefebvre et al 2001). Endurance conditioning increases mitochondria and provides more aerobic supplied energy,

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52 reducing lactate production (H olloszy & Coyle 1984, Honig et al 1992). Endurance conditioned muscles also have a higher lactate capacity a nd increased membrane transport, further reducing blood lactate levels during recove ry (Gladden 2000). Florida mana tees may be more physically fit than the Antillean subspecies. Another possible explanati on for the observed differences between the manatee subspecies may be due to differences in follow time prior to capture. Upon examining lactate levels of several Everglad es captured manatees, Tripp et al (2007) show an association between follow time and lactate. An individual followe d for 15 minutes showed a lactate level of 17 mmol/L whereas an individual followed for 50 minutes showed a lactate leve l of 23 mmol/L. It is possible that Antillean manatees in Belize an d Puerto Rico were followed longer than animals in Florida. Abnormal Individuals During this study we observed two abnorm a l individuals, CTB051 and TNP029. The result of monitoring the OT, HR, and RR of these individuals depicts the benefits and limitations of the field monitoring protocol. A capture on 05 January 2006 of CTB051, a 231 cm long male, captured in Tampa Bay, demonstrated a pronounced persistent bradycardia, with irregular rhythm, an instantaneous HR of 42 bpm, and a mean of 48 bpm by the ECG. HR was taken by stethoscope seven times during a 5 minute period and ranged between 28 bpm to 48 bpm. RR during this time was 4 breaths/5 minutes. Thirty minutes later, the HR stabili zed at 60 bpm, RR was 6 breaths/5 minutes. Oral temperature ranged from 34.1C to 34.6C. The SAA was 83 ug/mL. Additional care was taken in the field, once a bradycardia was recognized. This included alerting of symptoms to all involved field staff, more care in handling, induction of respir ations, and expediency in the

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53 overall health assessment. It is possible that th ese additional measures of care, helped to prevent the manatee from becoming further compromised. In the case of TNP029, an animal with >1200 ug/mL, no obvious signs of illness were apparent upon capture. However, the animal was found dead 32 days later from a chronic infection due to a watercraft impact relate d injury. At necropsy, pyothorax was confirmed (Florida Fish and Wildlife C onservation Commission unpublished da ta). The manatees OT was 34.7C; HR ranged from 52 bpm to 60 bpm; a nd RR ranged from 2 breaths/5 minutes to 15 breaths/5 minutes. Not all illne sses affect OT, HR, and RR to noticeable, abnormal values. This case demonstrates the limitations of OT, HR, and RR when monitoring the manatee. Future Monitoring Considerations For researchers capturing and handling m anat ees in the field, unders tanding the dynamics of manatee vital signs specific to their circumstances, and recogni zing abnormal trends in values overtime are essential for proper care of indivi duals. The effects of struggle and follow time should be studied further to improve manatee safe ty during capture procedures. Study of heat loss from the manatee fluke or other areas of the body, via thermography, may prove useful in further assessing thermal regulation by manatees in the field. HR of manatees should be monitored by stethoscope to bett er assess potential arrhythmias that might put an animal at increased risk. Manatees being captured may have significantly reduced breath holding ability, and should be allowed to frequently surface for ai r to prevent drowning. Quality of respirations should be studied with a capnograph, to enhance our understanding of manat ee capture recovery. Field use of a portable blood analyzer may help to further assist in assess ing the blood gas status of these animals. This study supports current post release behavioral a nd RR monitoring of field captured manatees, as physical recovery may still be occurring following a capture event. Establishing an historical record of indivi dual manatees OT, HR, and RR during long term

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54 studies is recommended to support h ealth assessments. Further st udy of the Antillean manatee is recommended in order to better understand physiological differences compared to Florida manatees. The OT, HR, and RR values and dynamics established in this study should serve as a point of reference for those individuals tasked with monitoring vital signs. For the safety of the animal, interpretation of results should be perfor med by an experienced manatee veterinarian or biologist.

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55 Table 3-1. Apparently healthy, free-ranging mana tees monitored during captures from 2004 to 2006 Juvenile 206-265 cm Adult >265 cm Location Male Female Male Female Total Florida 9 5 13 11 38 Belize 8 15 6 6 35 Puerto Rico 5 4 1 3 13 Total 22 24 20 20 86 14 calves (<206 cm) captured during this time were not included as part of this study. Size classification is based on modi fication of a working document by the USGS Sirenia Project, 30 September 1994. Size classes are based on straight length measurements. Table 3-2. Air and water temperatures (C) associated with manatees sampled for oral temperature, heart rate and respiration rate Belize and Puerto Rico Florida Mean (SD)) Median Range Mean (SD) MedianRange Air 30.7 (.9) 30.4 24.2-40.823.8(.0) 22.0 14.7-31.9 Water 28.9 (.5) 28.9 25.1-31.525.9(.2) 25.6 21.5-30.9

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56 Figure 3-1. OT, HR, and RR values in relation to invasive procedures that occurred to BZ05M79, a 225 cm male, on April 17, 2005. Manat ees in this study generally did not show extreme reactions in OT, HR, and RR as a dire ct result of a specific invasive procedure.

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57 ggg Time: Minutes 0102030405060 Oral Temperature C 31 32 33 34 35 36 Florida manatees; n=21 Antillean manatees; n=26 Figure 3-2. Mean oral temperatur es over time, with standard erro r bars, for healthy juvenile and adult manatees, collected immediately following capture Table 3-3. Descriptive statistics for oral temperatures of Florida and Antillean manatees at different time intervals during field monitoring Florida Manatees Antillean Manatees Time (min) n Mean (SD) Median Range Std. Error n Mean (SD) Median Range Std. Error 5 21 32.6 ( 1.8) 32.8 29.535.1 0.5 2634.6 ( 0.9) 34.9 32.636.0 0.3 15 21 34.1 ( 1.4) 34.5 29.835.7 0.3 2635.3 ( 0.6) 35.3 34.136.2 0.1 25 21 34.2 ( 1.4) 34.7 30.135.7 0.4 2635.2 ( 0.7) 35.3 33.636.2 0.1 40 21 34.3 ( 1.6) 34.9 30.735.8 0.5 2635.2 ( 1.6) 35.3 33.336.2 0.2 50 21 34.8 ( 1.5) 35.1 30.535.9 0.5 2635.2 ( 0.6) 35.25 34.036.1 0.1

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58 Time: minutes 0102030405060 Heart beats per minute 50 55 60 65 70 75 80 Florida manatee; n=25 Antillean manatee; n=22 Figure 3-3. Mean heart rates over time, with sta ndard error bars, for healthy juvenile and adult manatees, immediately following capture. Table 3-4. Descriptive statisti cs for heart rates of Florida an d Antillean manatees at different time intervals during field monitoring Florida Manatees Antillean Manatees Time (min) n Mean (SD) Median RangeStd.Error n Mean (SD) Median Range Std.Error 5 25 66 (.7) 70 48-80 2.7 2275 (.7) 74 64-88 2.4 15 25 62 (.1) 64 32-84 2.5 2167 (.4) 68 52-80 2.6 25 25 60 (.6) 57 40-80 2.9 2263 (.1) 63 44-80 2.3 40 25 59 (.7) 60 44-72 2.0 2263 (.9) 63 44-80 2.7 50 23 60 (.9) 62 45-68 2.2 2261 (.5) 60 48-84 2.5

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59 Time: Minutes 0102030405060 Respiration Rate 3 4 5 6 7 8 9 10 11 Florida Manatee; n=37 Antillean Manatee; n=48 Figure 3-4. Mean respiration rate s over time, with standard erro r bars, for healthy juvenile and adult manatees, immediately following capture. Table 3-5. Descriptive statistics for respiration rates of Florida and Antillean manatees at different time intervals during field monitoring Florida Manatees Antillean Manatees Time (min) n Mean (SD) Median RangeStd.ErrorTime (min) n Mean (SD) Median RangeStd.Error 5 37 6 ( 2.9) 6 1-13 0.5 5 489 ( 3.5) 9 3-17 0.5 15 37 6 ( 3.3) 6 0-17 0.5 15 487 ( 3.5) 8 2-15 0.5 25 37 6 ( 3.1) 5 0-17 0.5 25 486 ( 2.7) 6 0-11 0.4 40 37 5 ( 2.2) 5 1-10 0.4 40 486 ( 2.5) 5.5 2-12 0.4 50 36 4 ( 2.0) 4 0-8 0.4 50 485 ( 2.3) 5 2-12 0.4

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60 Figure 3-5. Correlation analyses of average heart rate versus aver age respiration for Florida and Antillean manatees. Pearson product moment correlation coefficient r2=0.856, p=0.00159; and r2=0.927, p=0.000112, respectively. Respiration Rate 4567891 0 Heart Rate 50 55 60 65 70 75 80 FL Heart Rate vs FL Respiration Rate Antillean Heart Ra te vs Antillean Respiration Rate Correlation Line y = 3.9915x + 38.915 r2 = 0.732 y = 4.6138x + 29.769 r2 = 0.8598

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61 Table 3-6. Initial heart rate re spiration rate and lactate of a female Antillean manatee BZ97F03, captured on 3 separate occasions during this study. Date captured Heart rate (beats per minute) Respiration rate (breaths/ 5 minutes) Lactate (mmol/L) 21 November 2004 92 16 27.5 16 April 2005 84 8 20 14 November 2005 60 3 9.7 Initial=within the fi rst 10 minutes of be ing secured upon capture Table 3-7. Mean times in minutes duri ng capture and handling of manatees Net Set to Secured Secured to Release Total time Location Mean ( SD) Range Mean ( SD) Range Mean ( SD) Range Florida 43.3 (.3) 4-271 57.4 (.3) 26-110 101.5 (.6) 35-311 Belize 43.0 (.4) 1-108 85.0 (.8) 42-125 129.7 (.3) 61-177 Puerto Rico 4.9 (.9) 3-6 55.6 (.1) 43-65 60.5 (.1) 48-69

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62 CHAPTER 4 MANATEE TEMPERATURE, HEART, AND RE SPIRATION MONITORI NG FIELD GUIDE The field guide in Appendix F describes mon itoring methods of oral temperature, heart rate, and respiration rate in mana tees, that can be used during fiel d rescues and research captures. The field guide was developed to serve as a usef ul point of reference for the newly introduced manatee biologist while monitoring vital signs of manatees in the field. The information collected from monitoring is intended to assist th e attending field veterinari an or senior biologist with decisions regarding individual animal care. Familiarization with medical terminology is suggested in order to enhance communication betw een veterinarians and bi ologists. Training in monitoring techniques from an expe rienced veterinarian or manatee biologist is recommended to ensure proper implementation.

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63 CHAPTER 5 CONCLUSION This thesis presents a norm al range of OT HR, and RR parameters of captured freeranging Florida (T. m. latirostris ) and Antillean ( T. m. manatus) manatees (Chapter 3). In so doing, it provides a successful methodology for monitoring HR and OT using electronic instrumentation (Chapter 2). The practical culmin ation of this research is a condensed OT, HR, and RR monitoring field guide for the field researcher (Chapter 4). Hypotheses Addressed Specific Aim 1: Establish tem perature, heart rate, and respiration rate parameters in captured manatees. Hypothesis 1a : Oral temperature will show a time dependent increase when ambient air temperature is above 26C. Yes, but the assumption that an increase in ai r temperature will cause more of a change in oral temperature is incorrect. We saw significant increases in oral temperature at ambient air values several degrees below 26C. Lower water temperature is likely more of a contributing initial factor affecting oral temperature in manate es. In the water, blood flow is reduced to the extremities by perivascular constriction, thereby reducing the loss of heat. When placed on land manatees will compensate by returning blood flow to their periphery as a thermoregulatory response. While the medium of air is 25 times le ss efficient at transferri ng heat than water, we saw no signs of overheating in captured manatees in a tropical climate. However, keeping animals shaded and wet when examined on land is still recommended as a precaution during hot sunny days. Hypothesis 1b: A healthy captured manatee's h eart rate is higher than 40 bpm. Yes, captured manatees generally initially presented heart rates well above the reported normal minimum of 40 bpm. A nor mal sustained HR for field captured manatees will be closer

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64 to 60 bpm. A downward trend in HR will generally occur over time, as the manatee recovers from exertion. Hypothesis 1c : Healthy captured manatees will show a normal respiration rate of 3-4 breaths/5 minute cycle (Bossart 2001). Yes, over time a healthy captured manatee will show a downward tr end of respirations within the normal range. However, initial respiration rates will be significantly higher than the reported normal, and it is not unusual for a rescued manatee to have sustained respirations above the normal range prior to release. The level of exertion from capture and handling on land is a significant factor for this phenomen on. Also, observed respirations on land are likely not of the same quality as those respirations in the water, given the strain of weight on the animal. Specific Aim 2: Determine possible correlations of oral temperature, heart rate, and respiration rate with blood chemistry. Hypothesis 2a : Oral temperature will show a time depe ndent increase in value after capture; heart rate and respiration rate will stabilize over time. No. Oral temperature stabili zed quickly during the monitoring period. However, heart rate and respiration rate did not always stabilize ove r time. Some animals showed elevated values upon release, which was likely due to rec overy time following capture struggle. Hypothesis 2b: A positive correlation exists between heart rate, potassium, and lactate concentration amongst hea lthy captured manatees. No. We did not establish a true correlati on for any monitoring parameter with a blood parameter. However, we established a signi ficant positive association between lactate and respiration rate. Elevated respiration rates can be a ge neral indicator of elevated lactate levels. The Captured Manatee and Future Monitoring Considerations If we consider the m anatee bradycardic re sponse while diving (Scholander & Irving 1941, Gallivan et al 1986), then a classic dive response is a very possible behavior for a distressed diving manatee. However, the common occurre nce and degree of a manatee dive response

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65 during free-ranging activity is de batable, given their considerab ly low metabolism and shallow water habitat (Scholander & Irving 1941, Hartman 1979, Butler 1982). The prevention of prolonged apnea by pouring water on the back of the manatees head to induce breathing, may also have prevented the occurrence of a dive response in this study. The manatee may also be more behaviorally ad apted to tolerate handling more than other animals. Their natural habit of becoming tidally stranded while foraging or being chased in a mating herd (Hartman 1979) has conditioned many populations with a behavioral and physiological response that is uni que to the shallow wate r habitats that they tend to inhabit. Manatee researchers have witnessed tidally strand ed individuals behaving in a sometimes playful manner, with normal HR, OT, and RR (Wong personal ob servations). It is likely that manatees are fully aware of the change in their surrounding s when they are pulled completely out of the water and placed on land. The inquisitive ma natee generally does not demonstrate an exceptional fight or flight response as hunted ungulates do, lacking the alarm communication that is characteristic of herd animals (Hartman 1979; Bateson & Bradshaw 1997). Many individual manatees, especially those in populous Florida, demonstrate not only a tolerance for people, but will often interact with swimmers (Reep & Bonde 2006). However, even given the manatees calm disposition, elevated heart and respiration rates were undoubtedly a consequence of the chase and struggle endured during capture. Differences in OT, HR, and RR between subspeci es were possibly a result of differences in environment, exertion, and physic al conditioning. The stable mean oral temperature of 35C shows adequate thermal regulati on, possibly due to excess heat lo ss occurring through the fluke (Rommel & Caplan 2003). The additional progr ession of HR and RR values toward reported normal values, between the subspecies of mana tees, suggests a physiological similarity.

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66 Monitoring of manatee OT, HR, and RR enables the field researcher, with a measurable framework, to further assess the immediate c ondition of this unique marine mammal. For common terrestrial animals vi tal signs monitoring has long been standard practice and commercial veterinary equipment has evolved to enhance a practitione rs monitoring ability (Bright 1997). Because manatee health assessment is not common practice, an effort should be made by the researchers to validate the use of instrumentation for this species. With some ingenuity many existing medical items developed fo r general human and veterinary medicine can be adapted for manatees. While we were unable to determine illness in TNP029 while in the field, abnormal OT, HR, and RR values can be appare nt in critically injured manatees For example, a critically injured boat strike adult manatee RNE0602, that died within 18 hrs from time of capture, had THR values during transport to Sea World which remained high and steady. OT was 36.6C, HR via ECG was generally at 73 bpm, and RR was between 5 breaths/5 minutes to 7 breaths/5 minutes (Florida Fish and Wildlife Conservati on Commission unpublished data). For two cold stress manatees, OT was initially 26.6C and increased only to 27.8C, and initially 28.3C and increased only to 30.6C (Florida Fish and Wildlife Comm ission unpublished data). Heart rates of these two cold stressed manatees ranged betwee n 40 bpm to 54 bpm, with varying qualities of breaths, and inconsistent RR ranging between 2 breaths/5minutes to 5 breaths/5 minutes. Additional improvements in current technologies applied in this study are recommended. A wireless thermometer transmitting to a hand held temperature display or lap top computer, would improve versatility. Assessing temperature regulation on other areas of the manatee, such as examining the body with a thermogra ph, would increase our understanding of thermoregulation. Heart rate monitoring could be improved with the use of a waterproof

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67 wireless ECG unit, transmitting to a lap top comput er. This would allow for digital data storage and increase field versatility. This study did not adequately addre ss quality of manatee respirations. A methodology to adequately meas ure tidal volume and use of a capnograph, in conjunction with OT, HR, and RR monitoring, would likely yield usef ul results for the clinician and researcher. A portable blood an alyzer should also be consider ed as an additional tool when assessing the condition of an animal, as we have seen that OT, HR, and RR monitoring is limited in its ability to serve as a predictor of changes in blood chemistry.

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68 1 2 3 4 5 6 7 8 APPENDIX A CAPTURE LOCATIONS Figure A-1. Map of m anatee capture locations

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69 Table A-1. Associated location descri ptions and coordinates for Figure A-1 Location description Coordinates Florida Latitude Longitude 1. Apollo Beach, Tampa: near TECO Big Bend power plant 27.01N 82.87W 2. Port of the Islands, Naples: residential and marina basin, Faka Union Canal 25.74N 81.03W 3. Everglades National Park: ar eas of Whitewater and Coot Bay 25.77N 80.37W Belize Latitude Longitude 4. Southern Lagoon: inshore waterway 17.21N 88.78W 5. Drowned Cays: off shore waters 17.25N 88.40W Puerto Rico Latitude Longitude 6. Boqueron: Rincn Canal 18.32N 67.15W 7. Guayanilla: Bahia de Guayanilla 17.49N 66.64W 8. Ceiba: near shore waters off former naval station Roosevelt Roads 18.40N 65.71W

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70 APPENDIX B BASIC MANATEE CAPTURE METHOD PHOTOS The basic m ethod of capturing a manatee using a seine net (Figures B-1 through B-3) was used in Puerto Rico, and was modified slightly to suit varying conditions in Florida and Belize. Figure B-1. Large seine net deployed from a cap ture boat (Photo Credit: Robert K. Bonde 2004)

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71 Figure B-2. Large seine net enclosed on a ma natee (Photo Credit: Robert K. Bonde 2004) Figure B-3. Manatee lifted onto a capture boat (Photo Credit: Robert K. Bonde 2004)

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72 Figure B-4. Land set near Florida s Teco Power Plant. In this manner, multiple manatees would be captured (Photo Credit: Chip J. Deutsch 2004)

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73 Figure B-5. A manatee capt ure in Belize. The manatee is co ntained within a small net and is being guided toward the stern of the boat by animal handlers. A surrounding large net is identified by the many buoys located in the background and the several buoys in the immediate foreground. (Phot o Credit: Yvonne Dartsch, 2005)

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74 APPENDIX C MANATEE LENGTH VERSUS WEIGHT Figure C-1. Length weight relati onship of m anatees captured in this study, from Florida and Belize. Not all animals were weighed, as weighing equipment was not available at every capture. Manatees from Puerto Rico are not included in the graph as the n=4 of available weights, is too low for analysis. Belize y = 20.257e 0.0108x R 2 = 0.9267 Florida y = 15.656e 0.0116x R 2 = 0.9331 0.00 100.00 200.00 300.00 400.00 500.00 600.00 700.00 0 50 100 150 200250300350 Straight Length (cm)Weight (kg)) Florida, n=34 Belize, n=14 Florida Belize

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75 APPENDIX D BASIC ECG RESULTS Table D-1. Results of ECG prin ted QRS tracing analyses for 25 of 47 heart rate m onitored manatees Animal Id Date Sex Straight length (cm)Instantaneous heart rate Rhythm BZ04F67 11/17/2004 F 219 79 Regular TTB115 12/14/2004 F 302 50 Regular TTB117 12/14/2004 F 277 51 Regular BZ97F03 4/16/2005 F 250 73 Regular BZ05F80 4/17/2005 F 234 51.4 Regular BZ05M79 4/17/2005 M 225 65.2 Regular BZ04M66 4/21/2005 M 294 49.1 Regular TPR28 4/29/2005 M 273 49.9 Regular TEP03 6/30/2005 F 246 69.8 Regular TEP01 6/30/2005 M 275 70.4 Regular BZ05M85 11/12/2005 M 245 67 Regular BZ05M85 11/12/2005 M 245 63.8 Regular BZ98M05 11/14/2005 M 235 54.5 Regular BZ05M87 11/15/2005 M 291 57.7 Regular BZ03F28 11/15/2005 F 214 77.1 Regular BZ05M93 11/18/2005 M 261 44.5 Regular TTB120 12/5/2005 M 259 57.5 Regular TTB119 12/5/2005 F 251 41 Regular TTB122 12/6/2005 F 276 68.5 Regular TTB123 12/6/2005 F 292 52.7 Regular TTB125 1/4/2006 M 277 64.7 Regular TTB127 1/4/2006 M 313 58.8 Regular TTB128 1/4/2006 M 296 71.4 Regular TTB128 1/4/2006 M 296 63.5 Regular TTB132 1/5/2006 F 302 53.6 Regular TTB101 1/5/2006 M 290 60.7 Regular CTB051 1/5/2006 M 231 42.7 Irregular .

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76APPENDIX E PLASMA CLINICAL BIOCHEMISTRY AND PROGESTERONE 4 VALUES Table E-1. Individual plasm a clinical biochemistry of 38 Florida ( Trichechus manatus latirostrus ) and 48 Antillean (T. m. manatus ) manatees concurrently monitored for oral te mperature, heart rate, and respiration rate Id Date Location Length (cm) ClassSex CK (U/L) Lactate (mmol/L) SAA (ug/mL) Na (mEq/L) K (mEq/L) Na/K CEP0501 06/28/05 Florida 220 2 F 8212 30 150 5.3 28.30189 CNP0401 01/17/04 Florida 245 2 M 7992.350 148 5 29.6 CTB046 12/14/04 Florida 212 2 F 33213 31 157 5.6 28.03571 CTB048 01/04/06 Florida 232 2 M 14117.5<10 150 4.5 33.33333 CTB049 01/05/06 Florida 223 2 M 19215.4120 152 5.2 29.23077 CTB051 01/05/06 Florida 231 2 M 1249 83 153 5.7 26.84211 TEP01 06/30/05 Florida 275 3 M 7618.9<10 152 5.9 25.76271 TEP02 06/30/05 Florida 284 3 M 8118.9<10 151 4.7 32.12766 TEP03 06/30/05 Florida 246 2 F 5111.718 158 4.8 32.91667 TEP04 07/01/05 Florida 311 3 F 13031 <10 150 5.3 28.30189 TEP05 12/01/05 Florida 275 3 M 29617 73 152 5.9 25.76271 TNP025 01/15/04 Florida 242 2 M 32311.9800 147 4.7 31.2766 TNP026 01/15/04 Florida 312 3 M 211<0.543 147 5.2 28.26923 TNP027 01/15/04 Florida 267 3 M 41320 360 149 4.3 34.65116 TNP028 01/16/04 Florida 234 2 M 57217 350 151 4.3 35.11628 TNP029 04/18/04 Florida 289 3 F 27813.2>1200 145 4.7 30.85106 TNP032 04/19/04 Florida 306 3 M 13116.2117 146 5.3 27.54717 TTB006 01/05/06 Florida 316 3 F 1617.8<10 152 5.6 27.14286 TTB069 12/16/04 Florida 299 3 M 10723.2<10 143 4.7 30.42553 TTB069 03/24/05 Florida 305 3 M 7711.512 146 4.7 31.06383 TTB081 12/14/04 Florida 300 3 F 1315.512 144 4.7 30.6383 TTB101 01/05/06 Florida 290 3 M 13819.4<10 153 4.7 32.55319 TTB109 12/14/04 Florida 295 3 F 1716.8<10 144 4.8 30 TTB115 12/14/04 Florida 302 3 F 10614.860 145 5.7 25.4386 TTB117 02/14/04 Florida 277 3 F 19412.2<10 143 5.2 27.5 TTB118 12/16/04 Florida 257 2 M 26314 <10 143 3.9 36.66667

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77Table E-1. Continued Id Date Location Length (cm) ClassSex CK (U/L) Lactate (mmol/L) SAA (ug/mL) Na (mEq/L) K (mEq/L) Na/K TTB119 12/05/05 Florida 251 2 F 1838.112 152 6.2 24.51613 TTB120 12/05/05 Florida 259 2 M 34211.111 152 5.2 29.23077 TTB120 12/05/05 Florida 259 2 M 34211.1 11 152 5.2 29.23077 TTB122 12/06/05 Florida 276 3 F 1935.5 11 153 5.8 26.37931 TTB123 12/06/05 Florida 292 3 F 1015 <10 156 5.1 30.58824 TTB124 12/06/05 Florida 285 3 M 13211 11 155 5.1 30.39216 TTB125 01/04/06 Florida 277 3 M 619.1 <10 146 4.9 29.79592 TTB126 01/04/06 Florida 256 2 M 10226.5 99 154 4.8 32.08333 TTB127 01/04/05 Florida 313 3 M 11623.8 <10 155 6.4 24.21875 TTB128 01/04/06 Florida 296 3 M 5720.5 <10 153 4.8 31.875 TTB130 01/04/06 Florida 278 3 F 8424.5 15 153 4.7 32.55319 TTB131 01/05/06 Florida 265 2 F 26614.8 19 149 4.6 32.3913 TTB132 01/05/05 Florida 302 3 F 9811.2 <10 154 6.3 24.44444 BZ00M13 04/16/05 Belize 297 3 M 7117.8 56 158 4.8 32.91667 BZ01M15 11/16/05 Belize 240 2 M 1139 <10 152 4.9 31.02041 BZ03F28 11/15/05 Belize 210 2 F 12014.5 <10 155 5.6 27.67857 BZ03F28 04/19/05 Belize 214 2 F 11417 59 163 5 32.6 BZ03F29 04/19/05 Belize 282 3 F 1186.6 <10 154 6 25.66667 BZ03F31 11/20/04 Belize 278 3 F 388 <10 136 4.8 28.33333 BZ03F31 04/18/05 Belize 280 3 F 8420.8 15 155 5.5 28.18182 BZ03F47 11/19/04 Belize 208 2 F 19318.5 61 117 4.2 27.85714 BZ04F67 11/17/04 Belize 219 2 F 13032 45 161 5.1 31.56863 BZ04F68 11/19/04 Belize 252 2 F 15829 28 130 4.7 27.65957 BZ04F69 11/20/04 Belize 285 3 F 10121.5 14 133 5.1 26.07843 BZ04F72 11/21/04 Belize 263 2 F 14737 <10 158 6.5 24.30769 BZ04F73 11/22/04 Belize 286 3 F 8124 <10 127 5.3 23.96226 BZ04F75 11/22/04 Belize 260 2 F 4631 <10 138 5.7 24.21053 BZ04M66 11/17/04 Belize 277 3 M 9335 <10 165 7 23.57143 BZ04M66 04/21/05 Belize 294 3 M 13717 <10 149 5.2 28.65385

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78Table E-1. Continued Id Date Location Length (cm) ClassSex CK (U/L) Lactate (mmol/L) SAA (ug/mL) Na (mEq/L) K (mEq/L) Na/K BZ04M71 11/21/04 Belize 250 2 M 10027 <10 159 5.4 29.44444 BZ04M74 11/22/04 Belize 280 3 M 7428 <10 147 5.8 25.34483 BZ04M74 04/16/05 Belize 283 3 M 11510 <10 154 4.8 32.08333 BZ04M77 11/22/04 Belize 256 2 M 16729.5 32 145 4.8 30.20833 BZ05F80 04/17/05 Belize 234 2 F 1499 23 149 4.9 30.40816 BZ05F81 04/17/05 Belize 212 2 F 18221 23 151 5.4 27.96296 BZ05F86 11/13/05 Belize 248 2 F 11813 24 157 5 31.4 BZ05F89 11/16/05 Belize 215 2 F 23324.576 157 5 31.4 BZ05F91 11/17/05 Belize 251 2 F 12622 34 156 5.3 29.43396 BZ05M79 04/17/05 Belize 225 2 M 8518.210 154 5.6 27.5 BZ05M85 11/12/05 Belize 245 2 M 15619.5<10 159 5.9 26.94915 BZ05M87 11/15/05 Belize 291 3 M 16822.579 131 4.1 31.95122 BZ05M88 11/16/05 Belize 279 3 M 9723.5<10 153 5.6 27.32143 BZ05M90 11/16/05 Belize 284 3 M 8913.5<10 153 4.7 32.55319 BZ05M93 11/18/05 Belize 261 2 M 8928.5<10 156 5 31.2 BZ97F03 11/21/04 Belize 246 2 F 6927.5<10 149 5.4 27.59259 BZ97F03 04/16/05 Belize 250 2 F 18320 <10 158 5.3 29.81132 BZ97F03 11/14/05 Belize 252 2 F 939.7<10 151 5.5 27.45455 BZ98M05 11/14/05 Belize 235 2 M 17723 13 155 4.6 33.69565 CPR0501 04/27/05 PuertoRico 222 2 M 22315.215 151 5.5 27.45455 TPR07 05/01/05 PuertoRico 297 2 M 6115.2<10 151 5.7 26.49123 TPR20 06/07/04 PuertoRico 256 3 F 3522N/A 190 153 4.7 32.55319 TPR21 06/07/04 PuertoRico 261 3 F 62N/A <10 151 5.2 29.03846 TPR22 06/08/04 PuertoRico 273 2 M 242N/A 88 146 5.2 28.07692 TPR23 06/10/04 PuertoRico 225 2 F 166N/A <10 149 5.6 26.60714 TPR27 04/28/05 PuertoRico 273 2 F 12328 14 153 5.5 27.81818 TPR28 04/29/05 PuertoRico 250 3 M 8220.2<10 150 4.9 30.61224 TPR29 04/29/05 PuertoRico 264 2 F 9521 19 158 6.1 25.90164 TPR30 04/29/05 PuertoRico 270 2 M 12624 <10 158 5.9 26.77966

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79Table E-1. Continued Id Date Location Length (cm) ClassSex CK (U/L) Lactate (mmol/L) SAA (ug/mL) Na (mEq/L) K (mEq/L) Na/K TPR31 04/30/05 PuertoRico 268 3 F 27729.516 204 5.7 35.78947 TPR32 05/01/05 PuertoRico 265 2 F 8818 16 158 5.8 27.24138 TPR33 05/02/05 PuertoRico 288 3 M 935 <10 163 5.6 29.10714

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80 Table E-2: Plasma clinical biochemistry descriptive st atistics from 38 Florida ( Trichechus manatus latirostrus ) and 48 Antillean (T. m. manatus ) fieldcaptured manatees Florida Mean(SD) MedianRange CK (U/L) 193() 135 51-799 K+ mEq/L 5.1(.6) 5.1 3.9-6.4 Na+ mEq/L 150.1(.2) 151 143-158 Na+/K+ 29.7(.1) 29.9 24.2-36.7 SAA (ug/mL) 97() 12 10-1200 Lactate (mmol/L) 14() 13 0.5-31 Antillean Mean(SD) MedianRange CK (U/L) 195() 117 38-3522 K+ mEq/L 5.3(.5) 5.3 4.1-7 Na+ mEq/L 152(.5) 153 117-204 Na+/K+ 28.8(.8) 28.1 12.2 SAA (ug/mL) 25() 10 10-190 Lactate (mmol/L) 21() 21 5-37

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81 Table E-3. Abnormal creatine kinase (CK) activity identified from 38 Florida (Trichechus manatus latirostrus ) and 48 Antillean (T. m. manatus ) fieldcaptured manatees Id Date CK(U/L) TNP028 1/16/04 572 CNP0401 1/17/04 799 TPR20 06/07/04 3522 Table E-4. Abnormal lactate values identified from 38 Florida (Trichechus manatus latirostrus ) and 48 Antillean ( T. m. manatus ) fieldcaptured manatees Id Date Lactate(mmol/l) TTB126 01/04/06 26.5 BZ04M71 11/21/04 27 BZ97F03 11/21/04 27.5 BZ04M74 11/22/04 28 TPR27 04/28/05 28 BZ05M93 11/18/05 28.5 BZ04F68 11/19/04 29 BZ04M77 11/22/04 29.5 TPR31 04/30/05 29.5 BZ04F75 11/22/04 31 TEP04 07/01/05 31 BZ04F67 11/17/04 32 BZ04M66 11/17/04 35 BZ04F72 11/21/04 37 Table E-5. Abnormal serum amyloid A (SAA) values identified from 38 Florida ( Trichechus manatus latirostrus ) and 48 Antillean (T. m. manatus ) fieldcaptured manatees Id Date SAA (ug/mL) TEP05 12/01/05 73 BZ05F89 11/16/05 76 BZ05M87 11/15/05 79 CTB051 01/05/06 83 TPR22 06/08/04 88 TTB126 01/04/06 99 TNP032 04/19/04 117 CTB049 01/05/06 120 TPR20 06/07/04 190 TNP028 01/16/04 350 TNP027 01/15/04 360 TNP025 01/15/04 800 TNP029 04/18/04 >1200

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82 Table E-6. Abnormal potassium (K+) values identified from 38 Florida ( Trichechus manatus latirostrus ) and 48 Antillean (T. m. manatus ) fieldcaptured manatees Id Date K+ (mEq/L) TPR29 04/29/05 6.1 TTB119 12/05/05 6.2 TTB132 01/05/05 6.3 TTB127 01/04/05 6.4 BZ04F72 11/21/04 6.5 BZ04M66 11/17/04 7

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83 Table E-7: Progesterone 4 values from blood serum of heart rate monitored female manatees, determined by chemiluminescent immunoassay (Tripp et al. 2006) Id Date Loc Class Sex P4 ng/mL Pregnancy Status BZ03F28 11/15/05 Belize 2 F 0.17 non-preg BZ03F28 04/19/05 Belize 2 F 0.08 non-preg BZ03F29 04/19/05 Belize 3 F 0.11 non-preg BZ04F67 11/17/04 Belize 2 F 0.07 non-preg BZ04F72 11/21/04 Belize 2 F 0.12 non-preg BZ05F80 04/17/05 Belize 2 F 0.07 non-preg BZ05F81 04/17/05 Belize 2 F 0.22 non-preg BZ05F86 11/13/05 Belize 2 F 0.03 non-preg BZ05F89 11/16/05 Belize 2 F 0.13 non-preg BZ05F91 11/17/05 Belize 2 F 0.03 non-preg BZ97F03 11/21/04 Belize 2 F 0.07 non-preg TEP03 06/30/05 Florida 2 F 0.035* non-preg TTB109 12/14/04 Florida 3 F 0.03 non-preg TTB117 02/14/04 Florida 3 F 0.41 non-preg TTB119 12/05/05 Florida 2 F 0.07 non-preg TTB122 12/06/05 Florida 3 F 0.05 non-preg TTB123 12/06/05 Florida 3 F 0.23 non-preg TTB130 01/04/06 Florida 3 F 1.31 probable TTB131 01/05/06 Florida 2 F 0.03 non-preg TTB132 01/05/05 Florida 3 F 0.72 non-preg *Value based on plasma in lithium heparin; CT B046 was not tested due to inadequate serum amount

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84 APPENDIX F MANATEE MONITORING FIELD GUIDE

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96 LIST OF REFERENCES Bateson, P., and E. L. Bradshaw. 1997. P hysiological effects of hunting red deer ( Cervus elaphus). Proceedings of the Royal Society B 264:1-8. Bayly, W. M., B. D. Grant, R. G. Breeze, and J. W. Kramer. 1983. The effects of maximal exercise on acidbase balance and arterial blood gas tension in thoroughbred horses. Pages 401-407 in D. H. Snow, S. G. B. Persson and R. J. Rose, editors. Equine Exercise Physiology. Granta Editions, Cambridge, England. Bayly, W. M., B. D. Grant, and R. C. Pearson. 1987. Lactate concentrations in thoroughbred horses following maximal exercise under field conditions. Pages 426 in J. R. Gillespie and N. E. Robinson, editors. Equine Exercise Physiology 2. ICEEP Publications, Davis, California, USA. Bossart, G. 2001. Manatees. Pages 939-60 in L. A. Dierauf and F. M. D. Gulland, editors. CRC Handbook of Marine Mammal Medicine. Second edition. CRC Press, Boca Raton, Florida, USA. Bossart, G., T. H. Reidarson, L. A. Dierauf, and D. A. Duffield. 2001. Clinical Pathology. Pages 383-400 in L. A. Dierauf and F. M. D. Gu lland, editors. CRC Handbook of Marine Mammal Medicine. Second edition. CRC Press, Boca Raton, Florida, USA. Bright, J. M. 1997. Monitoring vital signs in clin ical and research animal s. Current Separations 16:2. Buss, I. O., and A. Wallner. 1965. Body temperat ure of the African elephant. Journal of Mammalogy 46(1):104-107. Butler, P. J. 1982. Respiratory and cardiovascular control duri ng diving in birds and mammals. Journal of Experime ntal Biology 100:195-221. Convention on International Trade in Endange red Species of Wild Flora and Fauna. 2004. Appendix I . Accessed 02 June 2004. Deutsch, C. J., J. P. Reid, R. K. Bonde, D. E. Easton, H. I. Kochman, and T. J. OShea. 2003. Seasonal movements, migratory behavior, and si te fidelity of West Indian manatees along the Atlantic Coast of the United States. Journal of Wildlife Management 67(1). Farr, R., J. M. Montserrat, and D. Navajas. 2004. Noninvasive monitoring of respiratory mechanics during sleep. European Respiratory Journal 24:1052-1060.

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97 Franzmann, A. W., C. C. Schwar tz, and D. C. Johnson. 1984. Baseline body temperatures, heart rates, and respiratory rates of moose in Alaska. Journal of Wildlif e Diseases 20(4):333337. Gallivan, G. J. and R. C. Best. 1980. Metabo lism and respiration of the Amazonian manatee ( Trichechus inunguis ). Physiological Zoology 53(3):245-253. Gallivan, G. J., R. C. Best, and J. W. Ka nwisher. 1983. Temperature regulation in the Amazonian manatee ( Trichechus inunguis ). Physiological Zoology 56:255-262. Gallivan, G. J., J.W. Kanwisher, and R. C. Be st. 1986. Heart rates and gas exchange in the Amazonian manatee ( Trichechus inunguis ) in relation to diving. Journal of Comparative Physiology B 156:415-423. George, J. H. 1965. Electronic monitoring of vital signs. The American Journal of Nursing 65(2):68-71. Gladden, L. B. 2000. Muscle as a consumer of lactate. Medicine and Science in Sports and Exercise 32(4):764-771. Harr, K., J. Harvey, R. Bonde, D. Murphy, M. Lowe, M. Menchaca, E. Haubold, and R. FrancisFloyd. 2006. Comparison of methods used to diagnose generalized inflammatory disease in manatees ( Trichechus manatus latirostris ). Journal of Zoo and Wildlife Medicine 37(2):151-159. Hartman, D. S. 1979. Ecology a nd behavior of the manatee ( Trichechus manatus ) in Florida. The American Society of Mammologis ts. Special Publication No.5. 153 pp. Haubold, E. M., C. J. Deutsch, and C. Fonnesbeck. 2006. Final Biological Status Review of the Florida Manatee (Trichechus manatus latirostris). Florida Fish and Wildlife Conservation Commission-Fish and Wildlife Research Institute, St. Petersburg, Florida, USA. Harvey, J. W., K. E. Harr, D. Murphy, M. T. Wals h, E. J. Chittick, R. K. Bonde, M. G. Pate, C. J. Deutsch, H. H. Edwards, and E. M. Haubol d. 2007. Clinical biochemistry in healthy manatees ( Trichechus manatus latirostris ). Journal of Zoo and Wildlife Medicine 38(2):269-279. Holloszy, J. O., and E. F. Coyle. 1984. Adaptati ons of skeletal muscle to endurance exercise and their metabolic consequences. J ournal of Applied Physiology 56:831-838. Honig, C. R., R. J. Connet, and T. E. J. Gayeski. 1992. O2 transport and its interaction with metabolism: A systems view of aerobic capac ity. Medicine and Science in Sports and Exercise 24(1):47-53. International Union for Conservation of Nature and Natural Resources. 2007. Red List of Threatened Species . Accessed 18 December 2007.

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98 Irvine, A. B. 1983. Manatee metabolism and its influence on distribution in Florida. Biological Conservation 25:315-334. Kooyman, G. L. 1973. Respirator y adaptations in marine ma mmals. American Zoology 13:457468. Kooyman, G. L. 1985. Physiol ogy without restraint in diving mammals. Marine Mammal Science 1(2):166. Lefebvre, L. W., M. Marmontel, J. P. Reid, G. B. Rathbun, and D. P. Domning. 2001. Status and Biogeography of the West I ndian Manatee. Pages 424-474 in C. A. Woods and F. E. Sergile, editors. Biogeography of the West Indies, 2nd ed. CRC Press, Boca Raton, Florida, USA. Manire, C. A., C. J. Walsh, H. L. Rhinehart, D. E. Colbert, D. R. Noyes, and C. A. Luer. 2003. Alterations in blood and urine parame ters in two Florida manatees ( Trichechus manatus latirostris ) from simulated conditions of releas e following rehabilitation. Zoo Biology 22:103-120. Medway, W., M. L. Bruss, J. L. Bengtson, and D. J. Black. 1982. Blood chemistry of the West Indian manatee ( Trichechus manatus). Journal of Wildlif e Diseases 18(2):229-234. Medway, W., and J. R. Geraci. 1986. Clinic al Pathology of Marine Mammals. Pages 791-797 in M. E. Fowler, editor. Zoo and Wildlife Medicine. Second edition. W.B. Saunders Company, Philadelphia, Pennsylvania, USA. Montan, J., I., Marco, X. Manteca, J. Lpez, and S. Lavn. 2002. Delayed acute capture myopathy in three Roe deer. Journal of Veterinary Medicine Series A 49:(2)93. Murphy, D. 2003. Sirenia. Pages 476-482 in Fowler, M. E., and R. E. Miller, editors. Zoo and Wild Animal Medicine. Fifth edition. Saunders, St. Louis, Missouri, USA. Noren, S. R., V. Cuccurullo, and T. M. Williams 2004. The development of diving bradycardia in bottlenose dolphins (Tursiops truncatus ). Journal of Comparative Physiology B 174:139-147. OShea, T. J., G. B. Rathbun, E. D. Asper, and S. W. Searles. 1985. Tolerance of West Indian manatees to capture and handling. Biological Conservation 33:335-349. OShea, T. J., and C.A. Salisbury. 1991. Be lize-A last stronghold for manatees in the Caribbean. Oryx 25(3):156-164. Osofsky, S. A. 1997. A practical anesthesia monitoring protocol for free-ranging adult African elephants (Loxodonta africana). Journal of Wildlife Diseases 33(1):72-77. Parker, G. H. 1922. The breathing of the Florida manatee ( Trichechus latirostris ). Journal of Mammalogy 3(3):127-135.

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99 Reep, R. L., and R. K. Bonde. 2006. The Florida Manatee: biol ogy and conservation. University Press of Florida, Gainesville, USA. 189 pp. Robergs, R. A., and S. O. Roberts, editors. 1997. Exercise Physiology: Exercise, Performance, and Clinical Applications. Mosby-Year Book Inc., St. Louis, Missouri, USA. Rommel, S. A., and D. H. Caplan. 2003. Vascul ar adaptations for heat conservation in the tail of Florida manatees (Trichechus manatus latirostris ). Journal of Anatomy 202:343-353. Rose, R. J., D. R. Hodgson, T. B. Kelso, L. J. McCutcheon, T. A. Reid, W. M. Bayly, and P. D. Bollnick. 1988. Maximum O2 uptake, O2 debt and deficit and muscle metabolites in thoroughbred horses. Journal of Applied Physiology 64:781. Scholander, P. F, and L. Irving. 1941. Experiment al investigations on the respiration and diving of the Florida manatee. Journal of Cellu lar and Comparative P hysiology 17(2):169-191. Siegal-Willott, J., A. Estrada, R. K. Bonde, A. W. Wong, D. J. Estrada, and K. E. Harr. 2006. Electrocardiography in two s ubspecies of manatee ( Trichechus manatus latirostris and T. m. manatus ). Journal of Zoo and Wildlife Medicine 37(4):447-453. Spraker, T. R. 1993. Stress and capture myopathy in artiodactylids. Pages 481-488 in Fowler, M. E., editor. Zoo and Wildlife Medicine. Second edition. W.B. Saunders Company, Philadelphia, Pennsylvania, USA. Sweeney, J. C., and S. H. Ridgway. 1975. Proce dures for the clinical management of small cetaceans. Journal of the American Vete rinary Medical Associ ation 167(7):540-545. Tenney, S. M., and N. H. Hanover. 1958. Corre lative observations on the electrocardiogram and morphology of the heart of the Florida manatee. American Heart Journal 56(6):933938. Tripp, K., K. Harr, and J. Verstegen. 2006. Va lidation of a serum-based pregnancy diagnostic for the Florida manatee. Pages 44-45 in Proceedings of the 37th Conference of the International Association for Aquatic Anim al Medicine, 6-10 May 2006, Nassau, Bahamas. Tripp, K., K. Harr, and J. Verstegen. 2007. Evaluation of Stress Analyses in the Florida Manatee. Pages 153-155 in Proceedings of the 38th Conference of the International Association for Aquatic Animal Medicine, 5-9 May 2007, Lake Buena Vista, Florida, USA. U.S. Fish and Wildlife Service. 2004. Threatened and Endangered Species Database System.
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100 Walsh, M. T., and G. D. Bossart. 1999. Manatee Medicine. Pages 507-516 in Fowler, M. E., and R. E. Miller, editors. Zoo and Wild Animal Medicine: Current Therapy. Fourth edition. W.B. Saunders Company, Phila delphia, Pennsylvania, USA. Williams, T. M., D. Noren, P. Berry, J. A. Este s, C. Allison, and J. Kirtland. 1999. The diving physiology of bottlenose dolphins. The Jour nal of Experimental Biology 202:2763-2769. White, J. R., D. R. Harkness, R. E. Isaacks, and D. A. Duffield. 1976. Some studies on the blood of the Florida manatee, Trichechus manatus latirostris Comparative and Biochemical Physiology 55A:413-417.

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101 BIOGRAPHICAL SKETCH Arthur W illiam Wong was born in Brooklyn New Yo rk. He received a B.S. in biology at Southampton College, Long Island University in 2000. During his undergraduate studies he worked in animal husbandry and training at the Zeehondencreche Seal Rescue and Rehabilitation Center in the Netherlands, the New York Aquarium and Central Park Zoo. In his final year he studied aboard a sailing vessel in the Caribbean. After graduation Arthur pursued field research in Hawaii, Maine, and the Dry Tortugas. In 20 03, he was hired as a marine mammal biologist with the Florida Fish and Wildlife Conser vation Commission. Within months of his employment, the University of Florida accepted him as a graduate student. This thesis is a product of his time while working as a state biol ogist and simultaneously pursuing his masters degree. This thesis was supported by the Univers ity of Florida-College of Veterinary MedicineAquatic Animal Health Program, and the Flor ida Fish and Wildlife Conservation Commission.