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Metomidate Hydrochloride as a Sedative for Transportation of Selected Ornamental Fishes

Permanent Link: http://ufdc.ufl.edu/UFE0021771/00001

Material Information

Title: Metomidate Hydrochloride as a Sedative for Transportation of Selected Ornamental Fishes
Physical Description: 1 online resource (152 p.)
Language: english
Creator: Crosby, Tina
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: anesthetic, aquaculture, cortisol, fish, glucose, gourami, koi, marketability, metomidate, ornamental, sedative, stress, transportation
Fisheries and Aquatic Sciences -- Dissertations, Academic -- UF
Genre: Fisheries and Aquatic Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Transportation of fishes is stressful and may negatively affect their health and appearance as well as reduce their marketability (e.g., fin erosion, scale loss, and erythema) post-transport. The objective of the current study was to determine if sedation with metomidate hydrochloride during transportation of two species would reduce plasma cortisol and blood glucose levels as well as improve fish appearance, behavior, and activity level relative to control fish. Fishes were transported for approximately 24 hours via truck and domestic airline under typical shipping conditions, and blood was sampled at 0, 2, 6, and 12 hours post-transportation. Fish species used in these studies were koi, an ornamental variety of the common carp Cyprinus carpio (120 to 150 mm total length TL), and threespot gourami Trichogaster trichopterus (50 to 76 mm TL). The recommended metomidate dosage range for sedation is 0.1?1.0 mg/L. Based on pilot studies, metomidate hydrochloride concentrations tested for koi were: 0, 1.0, 2.0, 3.0, and 4.0 mg/L, and concentrations tested for gourami were: 0, 0.1, 0.2, 0.3, and 0.4 mg/L. At time 0 hour post-transportation, metomidate concentrations of 3.0 and 4.0 mg/L inhibited a rise in plasma cortisol levels compared to control koi. Also, plasma cortisol levels (34.8 ng/mL) and blood glucose levels (25.3 mg/dL) from baseline koi (i.e., not transported or exposed to metomidate) were similar to previously reported baseline plasma cortisol (5 to 85 ng/mL) and plasma glucose (20 to 120 mg/dL) levels for the species. In the koi study at time 0 hour post-transportation, there were no differences in koi blood glucose levels among all metomidate treatments. In contrast, in the gourami study, blood glucose levels of gourami transported with 0.1, 0.2, and 0.3 mg/L metomidate appeared to exhibit a non-significant, non-linear lowered trend compared to other concentrations tested. Additionally, in this study, the use of 0.4 mg/L metomidate was contraindicated. Gourami in this treatment had the highest mean blood glucose levels of all transported gourami, including the control, at times 0 and 2 hours post-transportation. There was no difference in the overall appearance of koi among transported fish at time 0 hour post-transportation; but, koi transported with 1.0, 2.0, and 3.0 mg/L metomidate were not different from baseline koi that were not transported. Similar to the koi results, in the gourami study, at time 0 hour post-transportation, there was no difference in the overall behavior among transported gourami. But, the behavior scores and percentage of sellable gourami transported with 0.3 mg/L metomidate were significantly lower than baseline gourami that were not transported. Therefore, the use of 0.3 mg/L metomidate may be contraindicated for transportation of this species. In this experiment for koi, a concentration of 3.0 mg/L metomidate during transportation inhibited a rise in plasma cortisol levels and prevented deterioration of appearance. For threespot gourami, concentrations of 0.1 and 0.2 mg/L metomidate during transportation appeared to inhibit an increase in blood glucose levels without affecting appearance or behavior. In conclusion, metomidate use during transportation for both species had beneficial effects and may be valuable to researchers for inhibition of the stress response.
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.
Statement of Responsibility: by Tina Crosby.
Thesis: Thesis (M.S.)--University of Florida, 2008.
Local: Adviser: Petty, Denise.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0021771:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021771/00001

Material Information

Title: Metomidate Hydrochloride as a Sedative for Transportation of Selected Ornamental Fishes
Physical Description: 1 online resource (152 p.)
Language: english
Creator: Crosby, Tina
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: anesthetic, aquaculture, cortisol, fish, glucose, gourami, koi, marketability, metomidate, ornamental, sedative, stress, transportation
Fisheries and Aquatic Sciences -- Dissertations, Academic -- UF
Genre: Fisheries and Aquatic Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Transportation of fishes is stressful and may negatively affect their health and appearance as well as reduce their marketability (e.g., fin erosion, scale loss, and erythema) post-transport. The objective of the current study was to determine if sedation with metomidate hydrochloride during transportation of two species would reduce plasma cortisol and blood glucose levels as well as improve fish appearance, behavior, and activity level relative to control fish. Fishes were transported for approximately 24 hours via truck and domestic airline under typical shipping conditions, and blood was sampled at 0, 2, 6, and 12 hours post-transportation. Fish species used in these studies were koi, an ornamental variety of the common carp Cyprinus carpio (120 to 150 mm total length TL), and threespot gourami Trichogaster trichopterus (50 to 76 mm TL). The recommended metomidate dosage range for sedation is 0.1?1.0 mg/L. Based on pilot studies, metomidate hydrochloride concentrations tested for koi were: 0, 1.0, 2.0, 3.0, and 4.0 mg/L, and concentrations tested for gourami were: 0, 0.1, 0.2, 0.3, and 0.4 mg/L. At time 0 hour post-transportation, metomidate concentrations of 3.0 and 4.0 mg/L inhibited a rise in plasma cortisol levels compared to control koi. Also, plasma cortisol levels (34.8 ng/mL) and blood glucose levels (25.3 mg/dL) from baseline koi (i.e., not transported or exposed to metomidate) were similar to previously reported baseline plasma cortisol (5 to 85 ng/mL) and plasma glucose (20 to 120 mg/dL) levels for the species. In the koi study at time 0 hour post-transportation, there were no differences in koi blood glucose levels among all metomidate treatments. In contrast, in the gourami study, blood glucose levels of gourami transported with 0.1, 0.2, and 0.3 mg/L metomidate appeared to exhibit a non-significant, non-linear lowered trend compared to other concentrations tested. Additionally, in this study, the use of 0.4 mg/L metomidate was contraindicated. Gourami in this treatment had the highest mean blood glucose levels of all transported gourami, including the control, at times 0 and 2 hours post-transportation. There was no difference in the overall appearance of koi among transported fish at time 0 hour post-transportation; but, koi transported with 1.0, 2.0, and 3.0 mg/L metomidate were not different from baseline koi that were not transported. Similar to the koi results, in the gourami study, at time 0 hour post-transportation, there was no difference in the overall behavior among transported gourami. But, the behavior scores and percentage of sellable gourami transported with 0.3 mg/L metomidate were significantly lower than baseline gourami that were not transported. Therefore, the use of 0.3 mg/L metomidate may be contraindicated for transportation of this species. In this experiment for koi, a concentration of 3.0 mg/L metomidate during transportation inhibited a rise in plasma cortisol levels and prevented deterioration of appearance. For threespot gourami, concentrations of 0.1 and 0.2 mg/L metomidate during transportation appeared to inhibit an increase in blood glucose levels without affecting appearance or behavior. In conclusion, metomidate use during transportation for both species had beneficial effects and may be valuable to researchers for inhibition of the stress response.
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.
Statement of Responsibility: by Tina Crosby.
Thesis: Thesis (M.S.)--University of Florida, 2008.
Local: Adviser: Petty, Denise.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0021771:00001


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1 METOMIDATE HYDROCHLORIDE AS A SEDATIVE FOR TRANSPORTATION OF SELECTED ORNAMENTAL FISHES By TINA CHRISTINE CROSBY A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF TH E REQUIREMENTS FO R THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2008

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2 2008 Tina Christine Crosby

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3 Dedicated to my parents for all their love and support over the many years.

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4 ACKNOWLEDGMENTS I thank the chair and m embers of my s upervisory committee for their guidance and support: B. Denise Petty, Roy P. E. Yanong, Je ffrey E. Hill, and Kathleen H. Hartman. I thank the Tropical Aquacu lture Laboratory, School of Forest Resources and Conservation, Program in Fisheries and Aquatic Sciences at the University of Florida for financial support. I also thank USDA-CSREES fo r funding; Syndel Laboratories Ltd., Qualicum Beach, Canada for providing AquacalmTM; and Florida Fish Farms Inc., Center Hill, Florida; 5-D Tropical, Inc., Plant City, Florida; and Segres t Farms, Gibsonton, Florida for their generous contributions. This research was conducted under Institutional Animal Care and Use Committee IACUC #E186. I thank Jamie Holloway, Paul Anders on, Jenney Kellogg, Matthew DiMaggio, and Jonathan Keener of the University of Florida, and Olanike Adeyemo of the University of Ibadan for their assistance. I thank Heather Hamlin of the University of Florida for assistance with the methodology and interpretation of the plasma cor tisol assay and Lou Guillette and David Julian of the, University of Florida, Department of Zoology for the kind use of their laboratories. I thank Meghan Brennan of the University of Florid a, Institute of Food and Agricultural Sciences (IFAS) Statistics Department. I thank Benjamin for all of his late-night support calls. His sense of humor kept me laughing and light-hearted throughout. I thank my mother for her strength, courage, and support over the years and my father for his support and sense of humor. Both of them helped me become the person I am today. I also thank my friends for their encouragement during the good times and especially dur ing the rough patches.

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5 I thank Bill Falls for his support and guidance. And lastly, I thank Craig A. Watson for giving me the opportunity to advance my education and pursue a career in fisheries and aquaculture.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ............................................................................................................... 4 LIST OF TABLES ...........................................................................................................................9 LIST OF FIGURES .......................................................................................................................11 ABSTRACT ...................................................................................................................... .............13 CHAP TER 1 INTRODUCTION .................................................................................................................. 15 2 LITERATURE REVIEW .......................................................................................................20 Transportation of Ornamental Fishes .....................................................................................20 Stress, Cortisol, and Glucose ..................................................................................................25 Anesthesia and Anesthetic Agents .......................................................................................... 28 Metomidate .................................................................................................................... .........34 Koi ..........................................................................................................................................36 Threespot Gourami ............................................................................................................. ....37 3 KOI PILOT STUDIES ........................................................................................................... 42 Objectives .................................................................................................................... ...........42 Koi Cortisol Assay Validation and Glucose Meter Evaluation .............................................. 42 Methods ..................................................................................................................................42 Experimental Design ....................................................................................................... 42 Simulated Transportation ................................................................................................44 Blood Collection and Blood Chemistry .......................................................................... 45 Results .....................................................................................................................................48 Discussion .................................................................................................................... ...........48 Koi Blood Glucose Pilot Study ...............................................................................................49 Methods ..................................................................................................................................50 Experimental Design ....................................................................................................... 50 Simulated Transportation ................................................................................................51 Blood Collection and Blood Chemistry .......................................................................... 51 Results .....................................................................................................................................52 Discussion .................................................................................................................... ...........52 Koi Metomidate Concentrati on Range Finding Pilot Study ...................................................52 Methods ..................................................................................................................................52 Experimental Design ....................................................................................................... 52 Observations .................................................................................................................. ..53 Results .....................................................................................................................................53 Discussion .................................................................................................................... ...........54

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7 Koi 48 Hour in High Dose Metomidate Pilot Study ............................................................... 54 Methods ..................................................................................................................................54 Experimental Design ....................................................................................................... 54 Simulated Transportation ................................................................................................54 Observations .................................................................................................................. ..54 Results .....................................................................................................................................55 Discussion .................................................................................................................... ...........55 Koi Plasma Cortisol and Blood Glucose Pilot Study ............................................................. 55 Methods ..................................................................................................................................55 Experimental Design ....................................................................................................... 55 Simulated Transportation ................................................................................................56 Blood Collection and Blood Chemistry .......................................................................... 56 Results .....................................................................................................................................56 Discussion .................................................................................................................... ...........57 Koi Fright Substance (Schreckstoff) Pilot Study .................................................................... 57 Methods ..................................................................................................................................57 Experimental Design ....................................................................................................... 57 Blood Collection and Blood Chemistry .......................................................................... 58 Results .....................................................................................................................................58 Discussion .................................................................................................................... ...........59 Pilot Studies Discussion .........................................................................................................59 4 KOI EXPERIMENT ...............................................................................................................67 Introduction .................................................................................................................. ...........67 Methods ..................................................................................................................................71 Experimental Design ....................................................................................................... 71 Transportation ................................................................................................................ ..73 Blood Collection and Blood Chemistry .......................................................................... 74 Appearance, Behavior, and Ac tivity Level Observations ............................................... 77 Shipping Water Physicochemistry .................................................................................. 78 Results .....................................................................................................................................78 Blood Chemistry ..............................................................................................................78 Appearance, Behavior, and Ac tivity Level Observations ............................................... 79 Shipping Water Physicochemistry .................................................................................. 79 Discussion .................................................................................................................... ...........80 5 THREESPOT GOURAMI PILOT STUDIES ........................................................................ 96 Objectives .................................................................................................................... ...........96 Gourami Metomidate Range Finding Pilot Study .................................................................. 96 Methods ..................................................................................................................................96 Experimental Design ....................................................................................................... 96 Simulated Transportation ................................................................................................97 Observations .................................................................................................................. ..98 Results .....................................................................................................................................98 Discussion .................................................................................................................... ...........99

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8 Gourami Blood Glucose Pilot Study ......................................................................................99 Methods ................................................................................................................................100 Experimental Design ..................................................................................................... 100 Simulated Transportation .............................................................................................. 100 Blood Collection and Blood Chemistry ........................................................................ 101 Results ...................................................................................................................................102 Discussion .................................................................................................................... .........102 6 THREESPOT GOURAMI EXPERIMENT .........................................................................105 Introduction .................................................................................................................. .........105 Methods ................................................................................................................................108 Experimental Design ..................................................................................................... 108 Transportation ................................................................................................................ 110 Blood Collection and Blood Glucose ............................................................................111 Appearance, Behavior, and Ac tivity Level Observations ............................................. 112 Shipping Water Physicochemistry ................................................................................ 113 Results ...................................................................................................................................113 Blood Glucose ...............................................................................................................113 Appearance, Behavior, and Ac tivity Level Observations ............................................. 114 Shipping Water Physicochemistry ................................................................................ 115 Discussion .................................................................................................................... .........115 7 EXPERIMENT DISCUSSION ............................................................................................127 APPENDIX A APPEARANCE, BEHAVIOR, AND AC TIVITY EVALUATION FORM ........................ 130 B KOI DATA ...........................................................................................................................131 C GOURAMI DATA ............................................................................................................... 136 LIST OF REFERENCES .............................................................................................................140 BIOGRAPHICAL SKETCH .......................................................................................................152

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9 LIST OF TABLES Table page 2-1 Number of doses, as per label, per U.S. dollar for commercially available anesthetic agents that induce sedation in fishes. ................................................................................. 40 2-2 Koi transportation densities in a full-size, square-bottom plas tic shipping bag with 7.57 L water for up to 24 hours total transportation time. ................................................. 40 3-1 Observations of two koi placed into a 7.57 L tank with 3.78 L aerated well water .......... 61 3-2 Observations of koi stage of anesthesia after 48-hour sim ulated transportation with metomidate exposure and observation s of recovery post-exposure .................................. 62 4-1 Criteria for evaluation of koi m arketability based on appearance, behavior, and activity level obs ervations post-transportation. ................................................................. 87 4-2 Mean (SD) koi shipping water physicoc hem istry parameters. Different letters indicate significant differences across rows ( P < 0.05). .....................................................88 5-1 Gourami range finding pilot study observations .............................................................. 103 6-1 Evaluations of gourami appearance, behavi or, and activity level for observations of fish m arketability post-transportation. ............................................................................. 119 6-2 Mean (SD) gourami shipping water physicoc hem istry parameters. Different letters indicate significant differences across rows ( P < 0.05). ...................................................120 B-1 Mean (SD) koi plasma cortisol levels in ng/m L. Different letters indicate significant differences down columns (P < 0.05). ...........................................................132 B-2 Mean (SD) koi blood glucose levels in mg /dL. Different letter s indicate significant differences down columns (P < 0.05). ............................................................................. 132 B-3 Mean (SD) koi appearance sc ores. Different letters indi cate significant differences across rows (P < 0.05). ..................................................................................................... 133 B-4 Mean (SD) percentage of sellabl e tanks of koi based on appearance. ........................... 133 B-5 Mean (SD) koi behavior scores. .................................................................................... 134 B-6 Mean (SD) percentage of sellabl e tanks of koi based on behavior. ............................... 134 B-7 Mean (SD) koi activity level score s. Different letters indicate significant differences across rows (P < 0.05). .................................................................................. 135 C-1 Mean (SD) gourami blood glucose levels in m g/dL. Different letters indicate significant differences down columns (P < 0.05). ...........................................................137

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10 C-2 Mean (SD) gourami appearance scores. ........................................................................137 C-3 Mean (SD) percentage of sellable tanks of gouram i base d on appearance. Different letters indicate significant differences acr oss rows (P < 0.05). ....................................... 138 C-4 Mean (SD) gourami behavior scores. Different letters i ndicate significant differences across rows (P < 0.05). .................................................................................. 138 C-5 Mean (SD) percentage of sellable tanks of gouram i based on behavior. Different letters indicate significant differences acr oss rows (P < 0.05). ....................................... 139 C-6 Mean (SD) gourami activity level scores. .....................................................................139

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11 LIST OF FIGURES page 1-1 Typical shipping box with side cut away to show full-size, square-bottom plastic bag, polystyrene shipping box, and outer ca rdboard box used for transportation. ............ 18 1-2 Koi Cyprinus carpio Photo credit: Blackwater Creek Koi Farm s, Inc. ............................ 19 1-3 Threespot gourami Trichogaster trichopterus .................................................................. 19 2-1 On the left is a plastic, full-size, square-bo ttom shipping bag filled with 7.57 L of water and on the right are various sizes and shapes of plastic shipping bags .................... 41 2-2 Chemical structure of metomidate. .................................................................................... 41 3-1 Simulated transportation fish shipping box arrangement .................................................. 63 3-2 Koi plasma cortisol levels from cor tisol validation pilot study post sim ulated transportation. ....................................................................................................................63 3-3 Koi blood glucose levels from the cor tisol validation pilot study post sim ulated transportation. ....................................................................................................................64 3-4 Koi blood glucose levels from the bl ood glucose pilot study. T he vertical line separates baseline koi from koi exposed to simulated transportation. ...............................64 3-5 Koi plasma cortisol levels from the pl asm a cortisol and bl ood glucose pilot study after 24 hours in simulated transportation ......................................................................... 65 3-6 Koi blood glucose levels ( N = 4 koi per treatm ent) from the plasma cortisol and blood glucose pilot study following 24 hours in simulated transportation ........................ 65 3-7 Koi plasma cortisol levels from Schr eckstoff substance pilot study. ................................66 3-8 Koi blood glucose levels from Sc hreckstoff substance pilot study. ..................................66 4-1 Mean plasma cortisol levels of trans ported koi sam pled at times 0, 2, 6, and 12 hours post-transportation. ............................................................................................................89 4-2 Mean blood glucose levels of transpor ted koi sampled at tim es 0, 2, 6, and 12 hours post-transportation. ............................................................................................................90 4-3 Koi appearance scores ( N = 25 koi observed for each m etomidate treatment per observation time) from time 0 to 12 h ours and 7 days post-transportation .......................91 4-4 Koi appearance as a percentage of occurr ence of sellable versus unsellable tanks of fi sh ( N = 25 koi observed for each treat ment per observation time). ................................ 92

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12 4-5 Koi behavior scores (N = 25 koi observed for each m etomidate treatment per observation time) from time 0 to 12 h ours and 7 days post-transportation .......................93 4-6 Koi behavior as a percentage of occurrence of sellable ve rsus unsellable tanks of fish ( N = 25 koi observed for each treat m ent per observation time) ........................................ 94 4-7 Koi activity scores ( N = 25 koi observed for each metomidate treatment per observation time) from time 0 to 12 h ours and 7 days post-transportation .......................95 5-1 Gourami blood glucose levels after 48 hours m etomidate exposure. Letters denote significantly different groupings ( P < 0.05) with standard error bars ............................. 104 6-1 Mean blood glucose levels of threes pot gouram i at times 0, 2, 6, and 12 hours posttransportation ...................................................................................................................121 6-2 Gourami appearance scores (N = 25 gouram i observed for each metomidate treatment per observation time) ....................................................................................... 122 6-3 Gourami appearance as a percentage of occurrence of sellable versus unsellable tanks of fish ( N = 25 gouram i observed for each treatment per observation time) ......... 123 6-4 Gourami behavior scores ( N = 25 gouram i observed for each metomidate treatment per observation time). ...................................................................................................... 124 6-5 Gourami behavior as a percentage of o ccurrence of sellable versus unsellable tanks of fish ( N = 25 gouram i observed for each treatment per observation time) ................... 125 6-6 Gourami activity scores ( N = 25 gouram i observed for each metomidate treatment per observation time) ....................................................................................................... 126

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13 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 METOMIDATE HYDROCHLORIDE AS A SEDATIVE FOR TRANSPORTATION OF SELECTED ORNAMENTAL FISHES By Tina Christine Crosby December 2008 Chair: B. Denise Petty Major Department: Fisherie s and Aquatic Sciences Transportation of fishes is stressful and may negatively affect their health and appearance as well as reduce their marketability (e.g., fin eros ion, scale loss, and erythema) post-transport. The objective of the current study was to determine if sedation with metomidate hydrochloride during transportation of two species would reduce plasma cortisol and blood glucose levels as well as improve fish appearance, be havior, and activity level relative to control fish. Fishes were transported for approximately 24 hours via truc k and domestic airline under typical shipping conditions, and blood was sampled at 0, 2, 6, and 12 hours post-transportation. Fish species used in these studies were koi, an orna mental variety of the common carp Cyprinus carpio (120 to 150 mm total length [TL]), and threespot gourami Trichogaster trichopterus (50 to 76 mm TL). The recommended metomidate dosage range for seda tion is 0.1.0 mg/L. Based on pilot studies, metomidate hydrochloride concentrations test ed for koi were: 0, 1.0, 2.0, 3.0, and 4.0 mg/L, and concentrations tested for gouram i were: 0, 0.1, 0.2, 0.3, and 0.4 mg/L. At time 0 hour post-transportation, metomida te concentrations of 3.0 and 4.0 mg/L inhibited a rise in plasma cortis ol levels compared to control koi Also, plasma cortisol levels (34.8 ng/mL) and blood glucose levels (25.3 mg/dL) from baseline koi (i.e., not transported or

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14 exposed to metomidate) were similar to previous ly reported baseline plasma cortisol (5 to 85 ng/mL) and plasma glucose (20 to 1 20 mg/dL) levels for the species. In the koi study at time 0 hour post-transportation, there were no differences in koi blood glucose levels among all metomidate treatmen ts. In contrast, in the gourami study, blood glucose levels of gourami tran sported with 0.1, 0.2, and 0.3 mg/L metomidate appeared to exhibit a non-significant, non-linear lowered trend compared to ot her concentrations tested. Additionally, in this stud y, the use of 0.4 mg/L metomidate was contraindicated. Gourami in this treatment had the highest mean blood glucose leve ls of all transported gourami, including the control, at times 0 and 2 hours post-transportation. There was no difference in the overall appearan ce of koi among transported fish at time 0 hour post-transportation; but, koi transported with 1.0, 2.0, and 3.0 mg/L metomidate were not different from baseline koi that were not transported. Similar to the ko i results, in the gourami study, at time 0 hour post-transportation, there wa s no difference in the overall behavior among transported gourami. But, the behavior scores and percentage of se llable gourami transported with 0.3 mg/L metomidate were significantly lower than baseline gourami that were not transported. Therefore, the use of 0.3 mg/L metomidate may be contraindicated for transportation of this species. In this experiment for koi, a concentrati on of 3.0 mg/L metomidate during transportation inhibited a rise in plasma cort isol levels and prevented dete rioration of appearance. For threespot gourami, concentrations of 0.1 a nd 0.2 mg/L metomidate during transportation appeared to inhibit an increase in blood glucose levels without aff ecting appearance or behavior. In conclusion, metomidate use during transporta tion for both species had beneficial effects and may be valuable to researcher s for inhibition of the stress response.

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15 CHAPTER 1 INTRODUCTION Aquaculture is an im portant contributor to Floridas economy with a reported farm-gate value of $75 million in a 2005 agricultural census conducted by the United States Department of Agriculture National Agricultural Statistics Service (USDA NASS 2006 ). The ornamental fishes industry comprises the largest component of the Florida aqua culture farm-gate value and produces over 44% of th e total (USDA NASS 2006). The value of ornamental fishes is directly affected by their marketability (appearance, behavior and activity level). Ma rketability of fishes may be affected by handling (e.g., trapping and netting) and physical abrasion that result in both scale loss and frayed fins prior to, during, and after transportation. Not only can transportati on affect marketability, but also survival. In the Florida industry, an acceptable percentage of mortality following transportation is less than or equal to 3% of the shipment (M. Poznania k, Segrest Farms, persona l communication). To minimize physical damage and death, fishes must be handled carefully throughout transportation (Ross and Ross 1999, Francis-Floyd 1995, Crosby et al. 2006a). Ornamental fishes from Florida are transported worl dwide (Wedemeyer 1996b) to wholesale facilities, reta il facilities, and hobbyists. Ornament al fishes in Florida are typically shipped in plastic bags that contain water and oxygen gas at a ratio of approximately 60% oxygen gas to 40% water by volume (Crosby et al. 2006b). The ratio of oxygen gas to water may vary by fish species and size of bag used. Th e plastic bag is then placed into a polystyrene shipping box which provides thermal protectio n (Ross and Ross 1999, Lim et al. 2003). Ice or heat packs may be used depending on the season and species of fish being shipped. The polystyrene boxes are then placed into a labele d outer cardboard box for transportation (Crosby et al. 2006a) (Figure 1-1).

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16 Transportation of fishes can be a substant ial stressor. Stress is defined as the physiological change that occurs in response to an imposed demand on an organism that aids in the maintenance of homeostasis (Barton 1997) Acute stressors such as handling and transportation cause significant increases in plas ma cortisol levels, a biological indicator of stress, as demonstrated in American shad Alosa sapidissima (Shrimpton et al. 2001), coho salmon Oncorhynchus kisutch (Avella et al. 199 1), rainbow trout Oncorhynchus mykiss (Woodward and Strange 1987, Pickeri ng and Pottinger 1989), brown trout Salmo trutta (Pickering and Pottinger 1989), hybrid striped bass Morone saxatilis x Morone chrysops (Davis and Griffin 2004), and Nile tilapia Oreochromis niloticus (Barcellos et al. 1999). When plasma cortisol levels in fishes ar e elevated, blood flow and pressu re are increased, oxygen demand and gill perfusion are increased, and hepatic gluconeogenesis is stimu lated (Norris and Hobbs 2006). Additionally, oxygen consumption can increas e up to 20% (Wedemeyer 1996a). These physiological adaptations increase the chances of fish survival (Wedemeyer 1996a). However, studies have indicated that even small increas es in plasma cortisol levels can have an immunosuppressive effect that may lead to an increased incidence of disease and mortality (Brown 1993, Wedemeyer 1996a). Even gentle hand ling of fishes is a significant stressor that may result in physiological changes such as an increase in plasma cortisol and blood glucose levels. There are a variety of anesthetic agents such as tricaine methane sulfonate (i.e., MS-222), quinaldine, metomidate hydrochlorid e, and clove oil that have been used in shipping water to alleviate transportation related stress; however, it is important to note that there are no drugs currently approved by the U. S. Food and Drug Administration (FDA) for transporting fishes. The shipping water may be treated such that the fishes are shipped under sedation, a stage of

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17 anesthesia. Crosby et al. ( 2006c) found that threespot gourami Trichogaster trichopterus subjected to a 4 minute handling stress and then returned to holding tanks for 2 hours containing either 0.8 mg/L metomidate, 5 mg/L quinaldine, 0.14 mg/L Hypno (Jungle Laboratories, Cibolo, Texas), a product containing quinaldine, or 60 mg/L Tricaine-STM (i.e., MS-222) (Western Chemical, Ferndale, Washington) had significantly lower plasma cortisol levels compared to control individuals and individuals exposed to 3 g/L salt (NaCl). However, there may be problems associated with the use of thes e anesthetic agents. For example, sedation with MS-222 or quinaldine may cause an initial excita tory response that results in increased plasma cortisol levels; and clove oil has a slow induction time (Barton and Peter 1982, Robertson et al. 1987, Ross and Ross 1999). Metomidate hydrochloride (hereafter referred to as metomidate) is a potentially important tool for transportation of ornamental fishes becau se of the reported reduction in plasma cortisol and blood glucose levels with its use. Ind eed, Kreiberg and Powe ll (1991) reported that metomidate sedation of Chinook salmon Oncorhynchus tshawytscha lowered glucocorticoid levels (e.g., cortisol, cortisone) compar ed to stressed control individuals. The goals of these studies were to inves tigate the hypothesis that transportation of ornamental fishes under metomidate sedation would 1) reduce plas ma cortisol and blood glucose levels and 2) improve overall marketability (i .e., appearance, behavior activity level) of transported fishes. These studies were designed to determine if the use of metomidate as a shipping water additive to induce sedation had an effect on 1) koi, an ornamental variety of the common carp Cyprinus carpio (Figure 1-2), plasma cortisol levels blood glucose levels, and marketability (i.e., overall physical appearance behavior, and activity level) post-transportation; and 2)

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18 threespot gourami (Figure 1-3) blood glucose levels and market ability (i.e., overall physical appearance, behavior, and activity level) post-transportation. In order to meet these objectives, pilot experiments were conducted to determine th e metomidate concentra tion required to induce sedation in each species. Figure 1-1. Typical shipping box wi th side cut away to show fu ll-size, square-bottom, plastic bag, polystyrene shipping box, and outer ca rdboard box used for transportation of ornamental fishes.

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19 Figure 1-2. Koi Cyprinus carpio Photo credit: Blackwater Creek Koi Farms, Inc. Figure 1-3. Threespot gourami Trichogaster trichopterus

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20 CHAPTER 2 LITERATURE REVIEW Transportation of Ornamental Fishes Ornam ental fishes from Florida are transported worldwide. Th e quality of the fishes after transportation is dependent on several factors. Th ese factors include the in itial health status of the fishes, the conditions under which they are coll ected and packed for transport, and the water quality parameters of the shipping water, es pecially dissolved oxyge n, temperature, total ammonia, carbon dioxide, and pH. Compromised fish health, improper fish packing, and poor shipping water quality can lead to disease and concomitant mortality, and ultimately decreased profitability for the seller. The first factor of transportation is the initial he alth status of the fishes. Transportation of unhealthy animals may result in increased mortal ity during transport or after arrival at the destination (Wedemeyer 1996a). Prior to transp ortation, fishes may be treated prophylactically with chemotherapeutants to increase post-tran sport survival (Lim et al. 2003, Crosby et al. 2006b). Ideally diagnostic tests should be pe rformed to identify and document specific pathogens before any treatment. The use of chemotherapeutants without an accurate disease diagnosis may increase production costs. In addition, inappropriate prophyl actic drug treatments may harm fishes. Moreover, inappropriate use of any antibiotic can increase microbial resistance (Khachatourians 1998, Cabello 2006). There are several factors that affect how fish es are packed and include time since last meal, packing materials (e.g., bags, boxes, etc.), packing density, and shipping water additives. A common practice is to withhold feed from the fi shes for 1 days prior to transport to allow the digestive tract to be purged (Wedemeyer 1996a, Lim et al. 2003) as digestion may increase oxygen consumption by up to 50% (Wedemeyer 1996a). This practice also aids in maintenance

PAGE 21

21 of shipping water quality by reducing carbon di oxide and waste production (Wedemeyer 1996a, Ross and Ross 1999, Lim et al. 2003). Furthermore, fishes may not survive the additional oxygen demand required to sustain basal metabolism due to increased oxygen consumption from digestion and transportation stress (Wedemeyer 1996a ). However, Olsen et al. (2008) concluded that Atlantic cod Gadus morhua fed prior to acute handling had plasma cortisol levels that returned to basal levels more rapidly than in control individuals not fe d for 3 days prior to experimentation. This data suggest s that feeding fishes prior to acu te stress may result in a rapid return to physiological pre-stress conditions. Ornamental fishes are typically transported in a square-botto m, plastic bag (Crosby et al. 2006b) (Figure 2-1). A square-bo ttom bag has a large surface area that allows for maximum gas exchange between the water a nd oxygen gas (Crosby et al. 20 06b). These bags do not have folded edges that may trap fishes such as pillow bags which have a single seamed edge (Crosby et al. 2006b). Square-bottom bags come in a variety of sizes, and the size used for shipping depends on the species and density of fishes being shipped (Figure 2-1). The bags are then positioned into polystyrene shipping boxes whic h are placed into cardboard outer boxes for labeling and additional thermal pr otection (Figure 1-1). Once th e fishes are packed, the boxes are transported primarily via trucks and planes for 24 hours but travel time may be longer or shorter depending on the final destination. Freight is a major component of the cost of transportation of ornamental fishes; therefore, fishes are densely packed into shipping bags w ith minimal water volume to reduce the overall freight weight of the shipment (Kaiser and Vine 1998, Lim et al. 2003). Ornamental fishes are frequently packed into bags with 40% wate r to 60% oxygen gas, but this ratio may vary depending on the species being transported (Cro sby et al. 2006b). Co mmon shipping densities

PAGE 22

22 for some of Floridas ornamental fishes ar e 400 small cyprinids (e.g., barbs ranging between 15.9 and 19.1 mm TL) or 300 medium characins (e.g., tetras ranging in size between 28.6 and 38.1 mm TL) per square-bottom, full-size, plastic shipping bag (431.8 x 431.8 mm) that contains 7.57 liters of water (Crosby et al. 2006b). Fishes or bag density may be determined by dividing the total number or total weight of fishes by the vol ume of water. Optimal densities are species specific and are affected by beha vioral requirements for physical space (Wedemeyer 1996a) and total length of time in transport (Lim et al. 200 3). Additionally, transportation densities may be limited by potential adverse changes in water physicochemistry parameters (Lim et al. 2003). Chemicals such as antimicrobials may be a dded to the shipping water to reduce bacterial load; anesthetics to redu ce physical abrasions, modi fy aggressive behavior, or slow the fishs metabolism (Wedemeyer 1996a, Ross and Ross 1999, Cr osby et al. 2006a); or salt (NaCl) to reduce any adverse physiological responses such as chloride imbalances or osmoregulatory dysfunction due to losses from diuresis, scale lo ss, or epithelial damage (Wedemeyer 1996a, Lim et al. 2003). Ionoregulatory disturbances account for many of the losses that occur within a week or two after transport (Wedemeyer 1996a, Lim et al. 2003); therefore, the addition of salt to shipping water is a common practice. Sodium ions from salt added to shipping water are available for exchange with hydrogen ions (H+) or ammonium ions (NH4 +) from the blood of fishes (Spotte 1979). In one study, white bass Morone chrysops transported for 5 hours and held in 5 g/L salt post-transportation increased survival to 93% compared to 30% in control individuals (Allyn et al 2001). In another study, Carniero an d Urbinati (2001) added salt at 0, 1, 3, or 6 g/L to shipping water and transported matrinxa Brycon cephalus for 4 hours. They found the use of 6 g/L salt resulted in no change in fish plasma cortisol, plasma chloride, or blood

PAGE 23

23 glucose levels. Although salt a ddition to transport water was us eful for the white bass and matrinxa, not all species of fish can tolerate salt at 5 g/L. For example, Lim et al. (2003) found that the guppy Poecilia reticulata had lower mortalities posttransportation when shipped with 1 g/L salt compared to 3 or 6 g/L salt. In addition to initial health status of the fishes and packing fact ors, there are several shipping water physicochemistry f actors that affect transporta tion such as dissolved oxygen, temperature, total ammonia, car bon dioxide, and pH. One of the most important considerations when transporting fishes is the dissolved oxygen of the shipping water. The dissolved oxygen in transport water declines as a consequence of resp iration. In addition, stress due to handling and transportation may increase oxygen consump tion up to 20% (Wedemeyer 1996a). Low dissolved oxygen concentrations l ead to respiratory stress, tiss ue hypoxia, and possible mortality (Wedemeyer 1996a). Therefore, oxygen gas is added to shipping bags where it passively diffuses into the water and main tains the dissolved oxygen ideally at or above saturation during transportation. Another consequence of respiration is th e production of carbon dioxide. As carbon dioxide levels accumulate, the pH of the ship ping water declines (Wedemeyer 1996a). In addition, carbon dioxide is highly soluble and can easily diffuse across the gills, lowering the blood pH (Moran et al. 2008). Th e resultant blood acidosis decreases the affinity of oxygen (O2) to bind with hemoglobin (Hb) by weakening the Hb-O2 bond (Wedemeyer 1996a). Tissues become hypoxic when shipping water pH is lowe r than 6.5 and carbon dioxide levels are greater than 30 mg/L (Wedemeyer 1996a, Ross and Ross 1999) which are common shipping water physicochemistry conditions. Indeed, Moran et al. (2008) reported that juvenile yellowtail

PAGE 24

24 kingfish Seriola lalandi had a 30% decrease in hemoglobin (Hb) concentration when exposed to simulated transport for 5 hours and 8 or 50 mg/L carbon dioxide. Another critical consideration in transportation of ornamental fishes is the maintenance of optimal shipping water temperature. Ornament al fishes include temperate, tropical, and warmwater fish species. The optimal temperat ure range for temperate fishes is 9C, for tropical fishes is 22C, and for warmwater fishes is 21C (Stoskopf 1993b, Avault 1996, Wedemeyer 1996a). It is important to note that these are not acceptable ranges for temperature fluctuation during transportation. Maintenance of temperature is often difficult during winter or summer temperature extremes (Lim et al. 2003). Consequently, the use of heat packs in the winter and ice packs in the summer aid in maintaining water temperature during transport (Lim et al. 2003, Crosby et al. 2006b). The effect of these temperature ai ds diminishes over time, so delays during transportation may have an adverse effect on bag water temperatures. The use of ice or heat packs is also dependent on the specie s of ornamental fish being shipped (Crosby et al. 2006b). The major metabolic waste product excr eted by fishes is total ammonia (NH3 + NH4 +) (Tucker 1993, Wedemeyer 1996). The ratio of unionized ammonia (NH3) to ammonium (NH4 +) is primarily dependent on the pH of the water; however, temperature, and to a lesser extent salinity, also affects this ratio. Unionized ammoni a, the portion of total ammonia that is more toxic than ammonium, increases as the pH and te mperature of the water increase, and increases as salinity decreases. Exposure to unionized a mmonia may lead to gill damage, ionic imbalance, hyperventilation, and hyperexcitability (Stoskopf 1993b, Wedemeyer 1996a, Ip et al. 2001). Unionized ammonia may also disrupt macromolecu lar function by bonding with protons, thereby increasing blood and intracellular pH (Walsh 1998) However, this does not occur often in

PAGE 25

25 transportation because at a pH less than or equa l to 7, over 99% of the total ammonia is present as the less toxic ammonium (Ip et al. 2001). Therefore, unionized ammonia is not a problem during transportation once pH drops below 7. During transportation the concentration of both total ammonia and carbon dioxide increases and pH decreases in shipping water (Crosby et al. 2006a). A high concentration of total ammonia in the water inhibits the release of ammonium via the Na+/NH4 + channel (Spotte 1979, Israeli-Weinstein and Kimmel 1998) This inhibition increases th e influx of ammonium across the gills consequen tly increasing plasma ammonium leve ls (Israeli-Weinstein and Kimmel 1998). Furthermore, ammonium stress can incr ease blood glucose levels in common carp Cyprinus carpio (Israeli-Weinstein and Kimmel 1998). Stress, Cortisol, and Glucose Handling and transportation of fishes induce the stress response. The stress response can be defined as the physiological change that occu rs to m aintain homeostasis and compensate for an imposed demand on an organism (Barton 1997). Stress in fishes results in primary (endocrine), secondary (blood and tissue), tertiary (whole-animal), or quaternary (population and ecosystem) responses (Wedemeyer 1996a). The dur ation as well as seve rity of the stressor affects the degree of the response (Barton 1997). The stress response initially serves to increase an organisms chance of survival by initiating th e fight or flight respons e (Barton 1997, Al-Kindi et al. 2000). However, a chronic stress re sponse may become maladaptive (Barton 1997). The immediate physiological response to a stressor is stimulation of the hypothalamic portion of the brain to release adrenocorticot ropic hormone (ACTH) (Wedemeyer 1996a, Barton 1997, Schreck et al. 1997). Adrenocorticotropic hormone stimulates the interrenal cells to produce cortisol, the primary steroid hormone synt hesized by the interrenal tissue in teleost fishes (Hazon and Balment 1998, Norris and Hobbs 2006). During the stress response, cortisol

PAGE 26

26 aids in maintenance of homeostasis through activ ation of the central ne rvous system (CNS), increase in blood pressure, reduction of the infl ammatory and immune reaction, and stimulation of glucose production (Bamberger et al. 1996). Elevat ed levels of plasma co rtisol are considered an indicator of stress (Al-Kindi et al. 2000). Plasma cortisol cl earance is dependent on a number of factors such as stress, species, and age; however, after an acute stressor, plasma cortisol levels generally return to basal levels within a day (Mommsen et al. 1999). Several factors play a role in the plasma cortisol res ponse including species (Mommsen et al. 1999, Pottinger et al. 2000), genetic makeup (Schreck 1990, Fevolden et al. 1994), gender (Leach and Taylor 1980), developmental stag e (Laidley and Leat herland 1988, Schreck 1990, Mommsen et al. 1999), prior handling and heal th history (Schreck 1990), stocking density (Ainsworth et al. 1985, Ruane and Komen 2003), feeding hist ory (Ruane et al. 2002), temperature (Davis 2004), and photoperiod (Almazan-R ueda et al. 2005). For example, Ruane et al. (2002) found that plasma cortisol levels si gnificantly increased afte r confinement stress in common carp fry fed an increased amount of feed prior to confinement co mpared to individuals fed less feed prior to confinement. In c ontrast, Olsen et al. ( 2002, 2005, 2008) reported that fishes fed prior to acute handli ng stress had lower plasma cortis ol levels compared to unfed control individuals. The differen ce between the two results may be due to species va riability. In addition, crowding fishes may increase plasma cor tisol levels. For instance, Rotllant and Tort (1997) found that doubling the stocking density of red porgy Pagrus pagrus in a holding tank resulted in significant elevations of plasma cortisol levels from less than 10 ng/mL in control fish to 50 ng/mL in crowded individuals. Similarly, plasma cortisol le vels increased from 30 ng/mL to 155 ng/mL in pre-smolt and smolt Atla ntic salmon crowded for one hour (Pankhurst et al. 2008). Pottinger (1998) and Ruane (2001) also reported that common carp exposed to net

PAGE 27

27 confinement had plasma cortisol levels of 217 ng/mL. Similarly, Tanck et al. (2000) reported plasma cortisol levels of 338 ng/mL in carp exposed to a 40 minute temperature shock. The baseline plasma cortisol levels of common carp under culture conditions are 5 ng/mL at 25C (Tanck 2000, Goos and Consten 2002). In contrast, plasma cortisol levels for a variety of stressed fishes usually range between 100 ng/mL (Barton and Iwama 1991, Sumpter 1997); however, in some species plasma cort isol levels of stressed fishes may be greater than 1000 ng/mL or as low as 5 ng/mL (Barton and Iwama 1991). Indeed Mazik et al. (1991) found that striped bass Morone saxatilis had plasma cortisol levels of 1700 ng/mL after 5 hours of transport. The time required to detect a significant elevati on in plasma cortisol levels is frequently observed to be 5 minutes after the onset of an acute stressor (Barton and Iwama 1991, Sumpter 1997, Pottinger 1998). It is important to note that in a study with carp, species unknown, Ilan and Yaron (1976) demonstrated that sampling blood by cardiac puncture within 4 minutes in unanesthetized carp did not elicit an appreciable rise in plasma cortisol levels. Increased plasma cortisol levels stimulat e glucose production and increase the livers capacity to produce glucose (Wedemeyer 1972, Sc hreck et al. 1997, Mommsen et al. 1999). The production of glucose is a secondary response to stress (Mazeaud et al. 1977). Glucose aids the response to a stressor by increasing gill perf usion and reducing blood clotting time; however, some deleterious effects of chronic glucos e production are lymphope nia and interrenal cell hypertrophy (Wedemeyer 1996a). Grutter and Pankhur st (2000) found that the blackeye thicklip wrasse Hemigymnus melapterus, a tropical reef fish, had increa sed blood glucose levels after two hours of handling and transportation. Similarly, Fagundes et al. (2008) observed increased blood glucose levels after 12 hours of transportation in spotted sorubim Pseudoplatystoma corruscans,

PAGE 28

28 a food, sport, and ornamental catfi sh in Brazil. Even a short 15 second exposure to air increased plasma glucose levels by 25% in Atlantic salmon (Fast et al. 2008). The blood glucose response is slower than th e plasma cortisol response (Carballo et al. 2005); however, hyperglycemia provides energy fo r the fight-or-flight response (Wedemeyer 1996a). Therefore, plasma glucose levels must in crease in circulation relatively quickly. It is important to note that blood glucos e concentration is also depende nt on several factors such as species, metabolic state, and developm ental stage (Mommsen et al. 1999). Groff and Zinki (1999) reporte d plasma glucose levels of fed common carp to be 30 mg/dL whereas Palmeiro et al. (2007) indi cated a broader range of 20 mg/dL. The difference in the ranges may be due to sensitivity of the analyses performed, in variation among individual fish, or in methodology of blood sampling. For example, for every hour at room temperature, the blood glucose level is decr eased approximately 10% by the erythrocytes through glycolysis (Evans and Duncan 2003). Howe ver, there is little appreciable difference between blood glucose and plasma glucose if the glucose level is analyzed within 30 minutes of blood collection (A. Rick Alleman, University of Florida, personal communication). Subsequently, blood glucose levels in this experi ment were analyzed immediately after sample collection to avoid a change in glucose concentration due to glyc olysis, and to compare koi blood glucose levels with published koi plasma glucose levels. Anesthesia and Anesthetic Agents While there are different types of anesthesia that include local, regional, and general (Steffey 1995), ornam ental fishes are transported under general anes thesia. General anesthesia affects the entire body by elicit ing a reversible depression of the CNS through prevention of nerve impulses (Summerfelt and Smith 1990, Steffe y 1995). The word anesthesia is derived from Greek meaning insensible or without fe eling (Steffey 1995). Use of an anesthetic

PAGE 29

29 agent results in a dose-dependent or exposure-related c ontinuum of responses; that is, stages of anesthesia (Steffey 1995). There are several schemes that describe th e different stages of anesthesia, but one commonly used scheme is Stoskopfs (1995) four stages of anesthesia in fishes. The first stage of anesthesia is sedation in which fishes may vary in the response to stimuli and motion and respiratory rate are reduced, but equilibrium remains normal. The second stage of anesthesia is narcosis in which fishes do not respond to stimuli and have loss of equilibrium. The third stage of anesthesia is light to surgical anesthesia in which there is a further de crease in respiratory rate and loss of muscle tone. The fourth stage of anes thesia is medullary collapse in which there is total loss of gill movement followed in several minutes by cardiac arrest. Sedation is recommended for transportation of most fishes (Summerfelt and Smith 1990). Anesthetic agents used in a limited concentratio n or for a limited duration induce a sedative stage of anesthesia in fishes (Stoskopf 1995). In contrast, anesthetic agents used at a higher concentration or longer duration induce a light to surg ical stage of anesthesia. Fishes in surgical anesthesia have a total loss of equilibrium and markedly reduced ventilation (Stoskopf 1995, Ross and Ross 1999, Stetter 2001). Induction of narcos is or light to surgical anesthesia should be avoided when transporting fishes because lo ss of equilibrium during transport may create localized areas of oxygen depletion within the shipping bag or may cause obligate air-breathing fishes to drown. There are three general phases of anesthesia: induction, main tenance, and recovery (Ross and Ross 1999). Fishes are typically induced via an immersion bath in which the anesthetic agent is absorbed across the gills where it rapidly enters the circulator y system (Summerfelt and Smith 1990, Ross and Ross 1999). The depth of anesthes ia is related to expos ure time and gill to

PAGE 30

30 body weight ratio, which is highl y variable among fishes (Coyl e et al. 2004). Induction of anesthesia may also be affected by biological factors such as species, metabolic rates, lipid content, size of the fishes, a nd health status of the fishes (Summerfelt and Smith 1990, Ross and Ross 1999, Coyle et al. 2004) and environmental f actors such as temperature (Stetter 2001), oxygen gas (O2) (Gilmour 1998, Nikinmaa and Salama 1998), and pH (Coyle et al. 2004). Ross and Ross (1999) reported that obligate air-breathing fishes prove a challenge to sedate because they have the ability to respire atmospheric oxygen. Ross and Ross (1999) theorized that this may lead to an increase in the induction time to sedation and may be overcome by increasing the drug concentration. However, Cros by et al. (2006c) did not observe increased induction times in sedation of threespot gourami, an obligate air-b reather, with metomida te, quinaldine, or Hypno (Jungle Laboratories, Cibolo, Texas), a co mmercial product containing quinaldine. If fishes are induced to enter the third stage of anesthesia quickly, cortisol release may be prevented (Summerfelt and Smith 1990, Barton and Iwama 1991, Ross and Ross 1999). The inhibition of cortisol may be a useful tool for limiting the effects of handling and transport. Strange and Schreck (1978) found th at induction of a light to surg ical stage of anesthesia in yearling Chinook salmon Oncorhynchus tshawytscha with MS-222 within 5 minutes prior to a handling stress suppressed an increase in plasma co rtisol level; however, if the fish did not enter a light to surgical anesthetic stage but were only sedated, a significant increase in plasma cortisol level was observed. There are various factors that may influence the selection of an anesthetic agent, and may include legality, cost, human health risks, biol ogical and environmental factors, dose response, induction time, and recovery time. Ideally the an esthetic agent used for sedation should induce quickly, be easily maintained, and have a qui ck recovery time (Ross and Ross 1999); however,

PAGE 31

31 selection may also be dependent on the procedur e being performed or the preference of the researcher (Summerfelt and Smith 1990, King et al. 2005). An important consideration when choosing an anesthetic is the legality of its intended use. There are no drugs currently approved for transportation of fishes; however, two drugs are approved by the United States Food and Drug Administration (FDA) for temporary immobilization of all finfishes, Finquel (A rgent Laboratories, Redmond, Washington) and Tricaine-STM (Western Chemical, Ferndale, Washi ngton) (FDA 2008), both of which contain tricaine methane sulfonate. However, there are other anesthetic agents used with ornamental fishes that are not FDA-approved ; examples include quinaldine sulfate, clove oil, benzocaine, and 2-phenoxyethanol (2-PE) (Coyle et al. 2004) Ano ther selection factor is cost. The followi ng anesthetic agents used at the lowest effective concentration labeled fo r sedation are (listed in order from least expensive to most expensive in U.S. dollars for commercially available products): quina ldine sulfate, 2-PE, benzocaine, Aqui-STM, metomidate, MS-222, and clove oil (Table 2-1). Additionally, potential human health risks must be also considered; for exam ple, 2-PE is irritating to skin and eyes with prolonged contact and is toxic to the kidney, nervous system, a nd liver (Summerfelt and Smith 1990). Tricaine methane sulfonate dust may irri tate the lungs through inhalation or the skin through absorption (Ross and Ross 1999). Also, Be rnstein et al. (1997) noted that chronic exposure to tricaine methan e sulfonate is retinotoxic. Finquel and Tricaine-STM are approved for use in Ictaluridae, Salmonidae, Esocidae, and Percidae, but their use may be affected by bi ological factors. Tric aine methane sulfonate is fat soluble and may require longe r induction and recovery times (e.g., return to normal state without behavioral side effects) in large fishes or gravid fe males with a higher lipid content

PAGE 32

32 (Ross and Ross 1999, Coyle et al. 2004). Additiona lly, MS-222 is associated with deleterious physiological effects such as hypoxia, erythroc yte swelling, and hyperglycemia (Ross and Ross 1999). Wedemeyer (1996a) also noted elevated oxygen consumption with the use of MS-222 at concentrations of 50 mg/L. The efficacy of MS-222 may also be affected by environmental factors. Sylvester and Holland ( 1982) reported that as temperatur e increased from 12C to 22C, the absorption rates of MS-222 increased in the rainbow trout Oncorhynchus mykiss the common carp, and the fathead minnow Pimephales promelas Tricaine methane sulfonate may induce an in itial excitatory response that results in plasma cortisol production and hyperexcitabil ity (Barton and Pete r 1982, Davis et al. 1982, Robertson et al. 1987). Another potential proble m with MS-222 is that it continues to accumulate in tissues over time during exposure even after equilibrium in the blood has been achieved (Treves-Brown 2000). Therefore, an initial dose of MS-222 may continue to be absorbed and potentially lead to ventilatory a rrest due to complete CNS depression (Ross and Ross 1999). It is important to note that for f oodfish exposed to MS-222 there is a 21-day withdrawal period after exposure before they may be used for human consumption (Stetter 2001, Coyle et al. 2004). Another commonly used anesthe tic is quinaldine sulfate; however, its use may also induce an initial excitatory response that results in plasma cortisol production (Barton and Peter 1982, Davis et al. 1982, Robertson et al. 1987). Small (2003) anesthetized channel catfish Ictalurus punctatus in quinaldine sulfate for 30 minutes a nd observed elevated plasma cortisol levels. Water physicochemistry parameters ma y also influence effectiveness of quinaldine sulfate. Lower water pH reduces the efficacy of quinaldine sulfate (Treves-Brown 2000, Coyle et al. 2004). At pH 7, 97.4% of quinaldine sulfate is available fo r absorption; however, at pH 5

PAGE 33

33 the chemical is in the ionized form (i.e., quina ldinium ion) which is poorly absorbed with only 27.5% of the chemical available for absorption (Treves-Brown 2000). Clove oil is another frequently used anesthe tic, but there are several disadvantages to its use as well. Woody et al. (2002) found anesthetic induction time was significantly longer as fish size increased when using clove oil at concen trations of 50 mg/L in adult sockeye salmon Oncorhynchus nerka Clove oil also induces a dose-dep endent response, and longer recovery times are required as dosage ra tes increase (Ross and Ross 1999, Coyl e et al. 2004). Waterstrat (1999) found that after 10 minut es exposure to concentrations of 100 mg/L clove oil, the behavior of fingerling channel ca tfish returned to normal after 4 minutes in recovery. However, channel catfish exposed to 150 mg/L clove oil required 10 minutes to recover normal behavior (Waterstrat 1999). Also, Park et al. (2008) re ported that recovery times increased from 31.3 seconds to 67.5 seconds as clove oil concentrations increased from 50 mg/L to 300 mg/L in kelp grouper Epinephelus bruneus The substantial difference in the dose response and recovery time between channel catfish and kelp grouper suggest s there is species vari ability to clove oil exposure. Environmental water physicochemistry parameters also affect efficacy of clove oil too. In the roach Rutilus rutilus Hoskonen and Pirhonen (2004) found that anesthetic induction time with clove oil was significan tly decreased at 20C (69 5 s) compared to 5C (266 31 s). Another anesthetic widely used is benzocaine wh ich is fat soluble, and as with its relative tricaine methane sulfonate, may require longer in duction and recovery times in large fishes or gravid females due to lipid content (Ross a nd Ross 1999, Coyle et al. 20 04). Also, Mattson and Riple (1989) reported that mortality increased in Atlantic cod exposed for 3 minutes to 75 mg/L benzocaine (50%) compared to 40 mg/L benz ocaine (0%). Temperature also affects effectiveness of benzocaine. The concentration of benzocaine required for sedation of striped

PAGE 34

34 bass within 3 minutes increased as temperat ure decreased, 55 mg/L benzocaine at 22C compared to 80 mg/L benzocaine at 11C (Gilderhus et al. 1991). Metomidate Metom idate (Figure 2-3) is an imidazole-ba sed organic, aromatic heterocyclic compound (Branson and Booth 1995). It is a hypnotic drug that has been used in birds, fishes, and mammals, including pigs and humans (Brans on and Booth 1995, Treves-Brown 2000). A hypnotic induces unconsciousness or sl eep; therefore, metomidate is effective for anesthesia but not analgesia (Branson and Booth 1995, Treves-Bro wn 2000). Metomidate is not FDA-approved for use with fishes in the United States, but has be en approved for use in Canada as an anesthetic in aquarium and non-food fishes in the order S iluriformes (i.e., catfish) and in the families Cyprinidae (e.g., carp, goldfish), Poeciliidae (e.g. live-bearers), Centra rchidae (e.g., sunfish, bass), Cichlidae (i.e., cichlids), and Pom acentridae (i.e., saltwater damselfish). The mechanism of metomidate activity has no t been specifically identified but it is believed to be similar to etomidate in physiochem ical and pharmacological properties (Hansen et al. 2003). Etomidate is an analogue of metomidate that has also been used as an anesthetic in fishes. Etomidate enhances -aminobutyric acid (GABA) inhibito ry pathways (Grant et al. 1999), the major inhibitory transm itter in the CNS that inhibits firing of action potentials, and has a functional role in control ling spinal and cerebellar reflex es (Steffey 1995). Etomidate is metabolized by hepatic (Lee 2007) and plasma este rases, and has a half-life of 2.6 hours in humans (Lexi-Comp, Inc. 2007). In a study of metomidate residue and clearance times, juvenile Atlantic salmon were exposed to 0, 0.1, and 0.5 mg/L metomidate for two hours at a water temperature of 8C (C. Kennedy, Simon Fraser University; J. Powell, S yndel Laboratories; and P. McKenzie, Heritage Salmon, unpublished data). Muscle tissue was collected for residue analysis immediately after

PAGE 35

35 metomidate exposure and again after 2, 4, 6, 24, and 48 hours in recovery tanks. The muscle tissue residue and clearance time of metomida te increased as metomidate concentration increased. However, all of the metomidate con centrations tested were cleared from muscle tissue after 24 hours of recovery in water without the drug. In another study Hansen et al. (2003) documented metomidate half-lif e times of 2.2 hours in turbot Scophthalmus maximus held at 18.0 0.3C and 5.7 hours in halibut Hippoglossus hippoglossus held at 10.3 0.4C. Studies of metomidate use in fishes have fre quently examined the inhibitory effect of the drug on plasma cortisol levels (Robertson et al 1988, Thomas and Robertson 1991, Iverson et al. 2003). Metomidate is believed to suppress 11hydroxylation of choles terol, part of the biochemical pathway required for the production of plasma cortisol in the interrenal tissue (Wada et al. 1988, Olsen et al. 1995). Kreibe rg and Powell (1991) found that Chinook salmon first sedated with metomidate and then exposed to a handling stress had significantly reduced plasma cortisol levels compared to similarly treated control individuals. In a density study, Davis and Griffin (2004) sedated differe nt groups of hybrid striped bass ( M. saxatilis x M. chrysops ) with one of the following: clove oil, Aqui-STM, metomidate, MS-222, quinaldine, and quinaldine sulfate; metomidate was the only sedativ e in which plasma cortisol and blood glucose levels were not significantly different from control individua ls. In addition, induction and recovery from metomidate seda tion is rapid (Ross and Ross 1999). Metomidate use may also reduce the respons e to stimuli. Juvenile black sea bass Centropristis striata anesthetized in clove oil or MS-222 had a reflexive response (i.e., tail movement) to caudal venipuncture, whereas the re sponse was not evident w ith metomidate usage (King et al. 2005). The lack of a response to stimu li is useful when collecting blood from fishes. However, in contrast to King et al. (2005), threespot gourami e xposed to 0.8 mg/L metomidate

PAGE 36

36 for 2 hours were very responsive to being ha ndled for blood collection (T. C. Crosby and coworkers, University of Florida, unpublished da ta). And, Hill noted that some rainbow sharks Epalzeorhynchos frenatum exposed to metomidate would interm ittently react strongly to stimuli (J. E. Hill, University of Florida, personal communication). Koi Koi (Figure 1-1) is a colorf ul variety of the common carp (Fam ily Cyprinidae) generally kept in ornamental ponds or aquaria for displa y (Cooper 1987). The Cyprin idae is the largest family of fishes (Billard 1999) with ov er 2,000 species (Moyle and Cech 2000), and is distinguished by a protrusile mouth, a toothle ss palate, tooth-bearing pharyngeal bones, and a keratinized pad on the roof of the pharynx (Bil lard 1999, Moyle and Cech 2000). The fish have a fusiform deep body with large eyes and l ack scales on their head (Cooper 1987, Moyle and Cech 2000). Koi and common carp originate in Asia (Cooper 1987, Moyle and Cech 2000, Barton 2007) and have an extensive natural geographic distribution (Lin and Peter 1991, Watson et al. 2004) that includes Eurasia, Japan, and the East Indian Islands (Lin and Peter 1991, Billard 1999, Hoole et al. 2001). Introduced populations are found in North Am erica as well (Lin and Peter 1991, Billard 1999, Hoole et al. 2001). Koi are tole rant of a wide temperature range (3C) (Cooper 1987, Moyle and Cech 2000) ; however, their optimal te mperature range is 16C (Hecker 1993). Cyprinid and koi culture have a long history. Cyprinid cultu re can be traced back to China and was described by Fan Li in the seventh century BC (Billard 1999). The culture of koi to develop different color patter ns originated in the Yamakoshi village of Niigata, Japan more than 200 years ago (Dennis 2007). There are currently 14 koi variety classifications recognized with over 100 color patterns (Hecker 1993).

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37 Koi belongs to the superorder Ostari ophysi, most members of which produce Schreckstoff substance (Pfeiffer 1982, Stoskopf 1995), a unique pherom one that is only released when the skin of the fish is damaged (Stos kopf 1995, Moyle and Cech 2000, Doving et al. 2005). Once released, the Schreckstoff substance is det ected by the olfactory or gans of conspecific, heterospecific, and closely related fishes. Exposure to Schreckstoff substance may elicit a variety of context-specific responses such as the fr ight response that is part of the flight response of predator avoidance (Pfieffer 1982, Sorensen and Caprio 1998, Barton 2007). In the natural environment the pheromone dissipates within a short time (Smith 1998). Koi is an economically important species in the U.S. that generated $6.5 million dollars in sales in 2005; koi cult ured in Florida represented 9% (i.e., $589,000) of the total koi farm-gate value for U.S. sales (USDA NASS 2006) The packing density of koi in a shipping bag is determined by the size of the fish that ar e transported. A commonly used shipping bag is a square-bottom, full-size, plas tic bag (393.7 x 368.3 x 571.5 mm) that contains 7.57 L water placed in a full-size shipping box (432 x 432 mm). The number of koi that may be packed into this size bag, as determined by fish size and trans ported for up to 24 hours, is listed in Table 2-2 (R. Slay, Florida Fish Farms, Inc., personal comm unication). However, these shipping densities may be adjusted depending on water temperature, time en route to destination, and method of transportation (R. Slay, personal communication). Threespot Gourami Threespot gouram i Trichogaster trichopterus (Family Osphronemidae) (Figure 1-2) (Rainboth 1996) is a small tropical freshwater aquarium fish native to southeastern Asia and the Indo-Malaysian archipelago (Axelrod et al. 1983 Petrovicky 1988). This small bodied fish can grow up to 150 mm in standard length (Pet rovicky 1988, Axelrod and Schultz 1990, Rainboth 1996) and has distinctly modified pelvic fins, similar in appearan ce to long, thin hairs used to

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38 sense the environment (Barton 2007 ). Threespot gourami are sexuall y dimorphic, and male fish possess a longer, more pointed dorsal fin that ex tends the length of the caudal peduncle (Cole et al. 1999). Threespot gourami is a carnivorous fish (Degani 1990, Graham 1997) with an optimal temperature range between 21C (Axelrod and Schultz 1990). These fish naturally live in wetlands such as marshes, swamps and canals, and frequently inhabit shallow, slow-moving or standing water with dense aquatic vegetation (Rainboth 1996, Cole et al. 1999). The gourami laterally migrates from permanent water bodies to flooded areas during the wet season and return to the permanent water bodies at the onset of the dry season (Rainboth 1996). Fishes in the Family Osphronemidae are obligate air breathers and may asphyxiate without access to the water su rface (Von Ramshorst 1981, Axelrod and Schultz 1990, Cole et al. 1999). Under normoxic conditions threespot gouram i acquire approximately 40% of their total oxygen consumption from the atmosphere (Bur ggren 1979, Graham 1997). This percentage increases under hypoxic conditions (Burggren 1979, Graham 1997). Threespot gourami thrive in low water levels that allow ease of access to th e surface for respiration. Although air above the water surface is utilized as a source of oxygen, gourami are stil l dependent on water for release of carbon dioxide and other gases (B urggren 1979, Graham 1997, Huang 2008). Fishes in the Suborder Anabantoidei, such as threespot gourami, are referred to as anabantoids. Anabantoids have a labyrinth organ that functions similar to a terrestrial lung and enables the fishes to respire air above the wate r surface (Stoskopf 1993a, Cole et al. 1999, Moyle and Cech 2000). The labyrinth organ allows the fishes to survive natu rally occurring hypoxic conditions (Huang et al. 2008). It is located above the gills and receives bl ood from the efferent artery of the first and second gills (Huang et al 2008). The labyrinth organ of threespot gourami

PAGE 39

39 consists of two highly convoluted suprabranchial chambers of the first gill arch (Graham 1997). Each suprabranchial chamber cons ists of three chambers with air entering the chambers via three openings (Graham 1997). Each opening can be closed off with a valve which allows aquatic respiration to occur (Graham 1997). Threespot gourami are biphasic air breathers (Graham 1997). Before air is inspired, biphasic air brea thers flush the labyrinth organ with a reverse stream of water that enters via the operculum; the organ is then refilled w ith freshly inspired air (Graham 1997). Ventilation with the labyrinth organ takes less than 0.5 sec, and it is emptied upon each exhalation (Burggren 1979, Graham 1997). The gill arches of Trichogaster species differ in morphology and function (Huang et al. 2008). The first and second gill arches regulate i ons, and the third and fourth gill arches have enlarged blood vessels and d ecreased numbers of filaments for faster transport of oxygen (Burggren 1979, Huang et al. 2008). This genus is economically important in Flor ida. In 2005, sales of tropical fishes, that include threespot gourami, were reported at a total of $32.4 million in 2005 (USDA NASS 2006). A large wholesale distri butor of ornamental fishes in Florida reportedly sells approximately 1 million Trichogaster sp. fish annually (wholesaler, personal communication). In Florida, medium-size gourami (i.e., rangi ng from 44.5.8 mm TL) are shipped 125 fish per square-bottom, full-size, plastic shipping bag with 7.57 L of water and large-size gourami (i.e., ranging from 50.8.5 mm TL) are shipped 75 fish per square-bottom, full-size, plastic shipping bag with 7.57 L of water (Crosby et al. 2006b).

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40 Table 2-1. Number of doses, as per label, per U.S. dollar for commercially available anesthetic agents that induce sedation in fishes. Anesthetic Agent Sedation Dosage (mg/L)Number of Doses per $1 Quinaldine sulfate 1.0 10,256 2-Phenoxyethanol 0.1.4 9,615 Benzocaine 10485 Aqui-STM 5.0 478 Metomidate 0.1.0 173 Tricaine-STM 10 153 Clove oil 50 96 Table 2-2. Koi transportation dens ities in a full-size, square-bo ttom, plastic shipping bag with 7.57 L water for up to 24 hours total transportation time. Size (mm) Number 76.5.0 25 152.4.2 10 228.6.0 5 279.4.6 2

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41 Figure 2-1. On the left is a plastic, full-size, square-bottom shipping bag filled with 7.57 L of water and on the right are various sizes and shapes of plastic shipping bags. Water colored for ease of viewing. Figure 2-2. Chemical structure of metomidate. C H CH3 N N C O CH3 O

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42 CHAPTER 3 KOI PILOT STUDIES Objectives A series of pilot studies was conducted with koi Cyprinus carpio to de termine 1) the validation of a plasma cortisol enzyme imm unoassay for use with koi, 2) the metomidate concentration required to induce sedation, 3) surv ivability in a 48 hour metomidate sedative bath, 4) the effect of metomidate sedation on plas ma cortisol and blood glucose levels following simulated transportation, and 5) the effect of blood collection and handling on plasma cortisol production in individuals in sepa rate tanks that share water. Koi Cortisol Assay Validation and Glucose Meter Evaluation A plasm a cortisol enzyme immunoassay was conducted to validate the use of the assay. In addition to the assay, an over-the-counter ha nd-held glucose meter was employed to determine blood glucose levels. Methods Experimental Design Koi selected for size in a range from 127 mm TL ( N = 60) were donated from a fish farm in Florida. Prior to e xperimentation, koi were held for two weeks in a recirculating tank system and fed once daily (Wardley TEN TOTAL ESSENTIAL NUTRITION Pond Pellets, Hartz Mountain Corporation, Secau cus, New Jersey) to satiation. The koi were maintained in a recirculating tank system that consisted of 30, 75.7-L tanks, a sump, a bubble-washed bead filter, an ultraviole t light sterilizer, and a fluidized bed filter that contained sand media. Each ta nk was filled with 37.8 L well water, had an air stone, and drained to a common sump. Koi were exposed to a 12 h light: 12 h dark photoperiod.

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43 Two koi were placed into each tank with polys tyrene partitions pl aced between tanks to reduce the potential stressful effect of visual s timuli from individuals in adjoining tanks. The tanks were covered with a lid made of plastic mesh and PVC tubing to prevent koi from jumping out of the tanks. An LDO digital meter (HQ20, Hach Company, Loveland, Colorado) was used to measure dissolved oxygen (DO), pH, and temperature data directly from each tank once a week. Immediately after recording the DO, pH, and temp erature a water sample was collected and total ammonia (NH3 + NH4 +), nitrite (NO2 -), alkalinity, hardness, sali nity, and carbon dioxide (CO2) were analyzed within an hour of collection usi ng a Hach Freshwater Fi sh Farmers Kit (FF-1A, Hach Company, Loveland, Colorado) and a salinity refractometer (Aquatic Eco-Systems, Inc., Apopka, Florida). The system was disinfected and refilled prio r to addition of the koi which did not allow time for the nitrifying bacteria to be established. Therefore, sa lt (NaCl, Morton Salt, Chicago, Illinois) was maintained in the system at 2.5.0 g/L. The salt was added to a 5-gal bucket with holes drilled into the bottom and sides. The bucket was placed above the water line on a submerged plastic crate in the sump. Water flowed into the bucket directly from the fluidized bed filter return line. The salt dissolved within 30 minutes. Water quality in the recirculating tank system was maintained at 0.3.0 mg/L total ammonia; unionized ammonia of 0.06.20 mg/L ; 0.17.18 mg/L nitrite; temperature of 24.2 24.8C; pH of 8.5.6; alkalinity of 51 mg/L; hardness of 136 mg/L; carbon dioxide of 5 mg/L; and dissolved oxygen of 7.1.6 mg/L. The unionized ammonia level was 0.20 mg/L wh ich is higher than the level that is considered potentially toxic (i .e., 0.05 mg/L) for fishes; however koi did not exhibit signs of

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44 ammonia toxicity, for example, hyperventilation and hyperexcitability (Wedemeyer 1996a). In addition, the nitrite level was above the level considered potentially to xic to fishes (i.e., 0.10 mg/L); however, koi were not observed to exhibit signs of nitrite toxicity for example, lethargy and crowding near aeration equipment or wate r outlets (Wedemeyer 1996a). Additionally, Lewis and Morris (1986) reported th e LC-50 for common carp due to nitrite to be greater than 40 mg/L in 96-hours; however, an increase in cort isol level may occur at much lower nitrite concentrations. Despite the pot entially toxic levels of unionized ammonia and nitrite, the adverse water quality did not appear to affect th e koi blood glucose or plas ma cortisol levels. The blood glucose levels were 12 mg/dL, similar to the plasma glucose levels reported for normal plasma biochemical reference interval s for common carp of 20 mg/dL (Palmeiro et al. 2007). Plasma cortisol levels were 4.6.9 ng/mL and were comparable to the baseline levels for carp of 5 ng/mL (Tanck 2000, Goos and Cons ten 2002). In order to reduce or prevent possible hypoxia due to methemoglobinemia from n itrite toxicity, salinity in the recirculating tank system was maintained with salt at 2.0.5 g/L for a ll subsequent experiments. Simulated Transportation Five koi were random ly chosen and exposed to simulated transportation for each of four treatment times: 0 hour, 30 min, 1 hour, and 4 hour s. There were only 2 people available to sample the koi and it was difficu lt to obtain blood from all five koi per tank within 4 minutes. Therefore, three koi from each tank were placed into a square-bottom, quarter-size, plastic shipping bag (191 mm x 165 mm x 500 mm) with 3.78 L aerated well water. Ten minutes later the remaining two koi were bagged. The bags were filled with oxygen gas, closed with a rubber band, and placed into polystyrene, square, single shipping boxes (432 x 432 mm) with a lid (Figure 1-1).

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45 The two shipping boxes were positioned on a w ood pallet with an air compressor (Sears Craftsman hp, 22.7-L, Hoffman Esta tes, Illinois) also placed on the pallet to simulate the movement of transportation (Figure 3-1). Th e air compressor shook the pallet for 30 seconds every 16 minutes for 4 hours, the duration of the experiment. In addition, a double piston air pump (Supreme Dynamaster air pu mp, Monroeville, Pennsylvania) was placed onto the top of each box to simulate the movement of transportation, and operated for the duration of the experiment. Blood Collection and Blood Chemistry At each sam ple time one of the bags was opened and the koi were placed in a bath containing 250 mg/L tricaine methane sulfona te (MS-222) and 500 mg/L sodium bicarbonate (NaHCO3) for 60 seconds. A new MS-222 sedative bath was prepared prior to each sample time interval. The koi were removed from the se dative bath and immediately placed into dorsal recumbency onto a wetted foam pad. Blood sample s were collected with a 25-G needle fitted on a 1-mL syringe containing approximately 0.06 mL sodium heparin (Baxter Healthcare Corporation, Deerfield, Illinois). The head and body of the koi were covered with a wet chamois cloth to restrain the animal and 0.5 mL blood was collected from the caudal vessels. Koi were sampled within 4 minutes from when a net first en tered the bag to standardize the sampling time. This time included the 60 second sedative bath. Ko i were then placed back into their original tank on the recirculating tank system after blood collection was completed. If 0.5 mL total volume blood and heparin was collected before the 4 minute time limit expired, the blood sample was immediately subm itted for blood glucose analysis. If 0.1.5 mL total volume was collected with in 4 minutes, blood glucose was analyzed from the collection attempt. However, if less than 0.1 mL tota l volume was collected or no blood was collected

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46 within four minutes, sampling ceased and the koi was placed back into the tank from which it originated. Blood glucose was immediately anal yzed using a hand-held Ascensia ContourTM glucose meter (Bayer Healthcare, Morristown, New Jersey). The meter required 0.6 L of blood, analyzed blood in 15 seconds, and had a detectable range of 10 mg/dL. The blood glucose levels were then correct ed for heparin dilution. The remaining blood in the syringe was placed into a 1.5 mL microcentrifuge tube and held in a refrigerator at 4C for no more than 2 hours prior to centrifugation. Blood glucose data were anal yzed using one-way ANOVA at = 0.05 followed by Tukeys HSD comparison of means in JMP 5.1 Statistical Discovery Software (SAS Institute, Cary, North Carolina). For blood gl ucose levels that were below the minimum level of detection for the meter (i.e., less than 10 mg/dL), a value of 5 mg/dL was arbitraril y assigned in order to determine the significance of blood glucose le vels among the various concentrations of metomidate. Whole blood was centrifuged (HN S-II, Dam on/IEC Divison, Needham, Massachusetts ) for 5 minutes at 2500 rpm. The plasma was pi petted into 1.5 mL microcentrifuge tubes and placed into a -80C freezer for 21 days until furt her analysis. Plasma cortisol levels were determined using a competitive plasma cortis ol enzyme immunoassay (EIA) (Cayman Chemical Company, Ann Arbor, Michigan) with a detection range of 7.9 pg/mL. To prepare the plasma samples for cortisol analysis the plasma samples were first thawed on ice. Ethyl ether was used to extrac t the lipophilic components from the hydrophilic components of the plasma. Extraction consiste d of adding 5 mL ethyl ether to each 50 L plasma sample. The solution then sat undist urbed for one minute followed by two minutes

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47 vigorous vortex mixing (Vortexer, VWR LabShop, Batavia, Illinois), an additional one minute undisturbed, and finally tw o minutes in a dry ice chilled methanol bath at or below -34.4C. The ethyl ether containing the lipophilic components fo r each sample was then decanted into a clean test tube and the ether was allowed to evaporate. The process was then repeated in the original test tubes with the pe llet of lipophilic and water soluble components. The air-dried tubes were then rehydrated a nd diluted 400 times with EIA buffer (Cayman Chemical Company, Ann Arbor, Michigan). The manufacturers methods were then followed. In brief, samples were pipetted into each well on a 96-well plate along with eight cortisol standards in duplicate and a series of plate control samples. The cortisol standards and control samples were used to generate the standard curv e for the plate. Once samples were pipetted on the plate, it was incubated in a refrigerator at 4C for no more than 18 hours. After incubation, developing reagent (Cayman Chemical Company, Ann Arbor, Michigan) was added to each well. The plate was then placed on an orbital shaker (Orbitron IITM, Boekel, Feasterville, Pennsylvania) and allowed to develop at room temperature for 90 minutes. After developing, it was read with a microplate spectrophotometer (Bio -Rad Benchmark Plus, Bio-Rad Technologies, Inc., Hercules, Califo rnia) at a wavelength of 412 nm. Plasma cortisol levels and the correlation coefficient between the serial dilution data curve and the standard curve of the kit were evaluated with the Cayman Chemical Company (Ann Arbor, Michigan) cortisol analysis tool. The plasma cortisol levels were then corrected for heparin dilution. Plasma cortisol data were analyzed using one-way ANOVA at =0.05 followed by Tukeys HSD comparison of means in JMP 5.1 St atistical Discovery Software (SAS Institute, Cary, North Carolina).

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48 Of the twenty attempted blood draws, only 14 were of sufficient quantity for blood glucose analysis. Successful blood draws were fr om the following time periods: three at time 0 hour, five at time 30 minutes, two at time 1 hour, and four at time 4 hours post-transportation. Of the 14 successful blood draws, only 10 ha d enough plasma volume for plasma cortisol analysis: two at time 0 hour, th ree at time 30 minutes, three at time 1 hour, and two at time 4 hours post-transportation. Results Serial dilutions of plasma cortisol from koi were parallel with the norm al standard curve for the plasma cortisol assay with a correlation coefficient of r = 0.998, and verified validation. Plasma cortisol levels at time 0 hour (6.2 ng/mL ) were significantly lowe r than at subsequent sample times 30 min, 1 hour, a nd 4 hours (198.5.4 ng/mL) (ANOVA: F = 12.04; df = 3, 6; P = 0.006) (Figure 3-2); however, there was no significant difference among sample times 30 min, 1 hour, and 4 hours. The blood glucose levels from samples coll ected at time 0 hour (17.0 mg/dL) and time 1 hour (20.5 mg/dL) were not significantly different from each other, but were significantly lower than levels from koi sampled at time 30 min (40.8 mg/dL) or time 4 hours (56.3 mg/dL) (ANOVA: F = 6.57; df = 3, 16; P = 0.0042) (Figure 3-3). However, three of the five blood glucose readings from koi sampled after 1 hour in simulated transportation were below the detectable range of the meter. Blood glucose levels from sampling times 30 min and 4 hours were not significantly different from each other. Discussion The standard curve of serial dilutions of koi plasm a cortisol wa s highly correlated to the standard curve of the assay; therefore, the assay was validat ed for analysis of koi plasma cortisol levels. The plasma cortisol levels that were out of the expected range indicated that the

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49 samples from stressed koi required further diluti on to bring the levels wi thin the range of the standard curve. Simulated transp ortation resulted in an increase in koi plasma cortisol levels, thus indicating a physiological response to shipping stress. A hematological reference for fed common carp lists the normal range for plasma glucose levels as 30 mg/dL (Groff and Zinki 1999) while a more recent reference indicates the range to be 20 mg/dL (Palmeiro et al. 2007). Of th e blood glucose levels th at were detectable by the meter, that is, 10 mg/dL, there were four glucose levels analyzed that were lower than levels previously reported for common carp. Both Wedemeyer et al. (1990) and Groff and Zinki (1999) report lowered plasma glucose levels in fish es that are fed less or starved. In this pilot study, not feeding the fish the day before the ex periment may have contributed to the lower blood glucose levels obtained. The inability to collect blood from some fi sh for analysis may have affected the evaluation of plasma cortisol a nd blood glucose levels; however, th e significant increase in both levels suggested that handling and simulate d transportation induced a physiological stress response. Therefore, the investigation into transportation sedatives that may inhibit these physiological changes in transported koi was va lidated. There were several minutes with no activity between sampling times; so, the ten minute time stagger be tween bagging koi was reduced to six minutes in the experiment. Koi Blood Glucose Pilot Study Koi were ex posed to simulated transportation at concentrations of metomidate within the recommended dosage to elicit sedation (0.05.0 mg/L ). Blood glucose levels were analyzed after 24 hours of simulated transp ortation and metomidate exposure.

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50 Methods Experimental Design The koi were the sam e as previously desc ribed on page 42 in Experimental Design. There were 14 days between the previous experime nt and this experiment. Fish were fed once daily (Wardley TEN, TOTAL ESSENTIAL NUTRITION Pond Pellets, Hartz Mountain Corporation, Secaucus, New Jersey) to satiation. Feed was withheld one day prior to and the day of experimentation. The koi were maintained in the same system and water quality was analyzed as described previously (pag es 42 Experimental Design). Water quality in the recirculating tank system was maintained at 0.3.4 mg/L total ammonia; unionized ammonia of 0.06.07 mg/L; nitrit e of 0.07.10 mg/L; pH of 8.2.6; temperature of 25.0.3C; alkalinity of 137 mg/L; carbon dioxide of 5 mg/L; and dissolved oxygen of 7.5.7 mg/L. The unionized ammonia and nitrite levels were higher than the levels that are considered potentially toxic for fishes; howe ver, the koi did not display any signs of toxicity. Koi were subjected to simulated transportati on conditions at metomidate concentrations of 0, 0.05, 0.1, 0.5, 0.75, or 1.0 mg/L. There were two fish per replicate and three replicates per treatment. In addition, there were three tanks with two koi in each that did not receive metomidate exposure or simulated transport and re presented a baseline treatment. A metomidate stock solution of 1 mg metomidate per 1 mL distilled water was used to dose 10 L shipping water in a square-bottom, ha lf-size, plastic shipping bag (406 mm x 203 mm x 558 mm) to the appropriate metomidate concentration. Each ba g with shipping water and metomidate treatment was then shaken vigorously for 30 seconds.

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51 Simulated Transportation The two koi from each tank were placed into square-bottom, quarter-size, plastic shipping bags (191 mm x 165 mm x 500 mm) w ith 3.78 L aerated well water for each of the metomidate concentrations and 2.5 g/L salt. A six minute time stagger between bagging each tank of koi allowed adequate time for blood sampling to be completed. The bags were then filled with oxygen gas, closed with a rubber band, and pl aced into polystyrene, square, single shipping boxes (432 x 432 mm) with lids (Figure 1-1). Five boxes were stacked in two separate st acks on a wood pallet (Figure 3-1). An air compressor (Sears Craftsman hp, 22.7-L, Hoffman Estates, Illinois) was also placed on the pallet to simulate movement of shipping (Figure 3-1). The air compressor shook the pallet for 30 minutes every 16 minutes for the duration of th e experiment. In addition, a double piston air pump (Supreme Dynamaster air pu mp, Monroeville, Pennsylvania) was placed onto the top of each stack of boxes and operated the duration of the experiment. Blood Collection and Blood Chemistry Twenty four hours after sim ulated transpor tation began; blood was sampled from the three baseline treatment tanks followed by th e koi in bags. The koi were removed and immediately placed into dorsal recumbency onto a wetted foam pad. Blood samples were collected with a 25-G needle fitted on a 1-mL syringe containing about 0.06 mL sodium heparin. The head and body of the koi were covered with a wet chamois cloth to re strain the animal and 0.5 mL blood was collected from the caudal vessels. Koi were placed back into their original tank on the recirculating tank syst em after sampling was completed. Blood glucose levels were analyzed as previously described (pag e 45 Blood Collection and Blood Chemistry).

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52 Results Six out of 42 blood samples were too low to register the blood gl ucose levels on the blood glucose m eter: four at baseline, one at 0.5 mg/L metomidate, and one at 1.0 mg/L metomidate. There was no st atistical difference in blood glucose levels (21.5.5 mg/dL) among metomidate concentrations of 0, 0.05, 0.1, 0.5, 0.75, and 1.0 mg/L (ANOVA: F = 2.07; df = 6, 35; P = 0.08) (Figure 3-4). Discussion This pilot study yielded several blood glucose levels that were too low to be analyzed by the glucose m eter (less than 10 mg/dL). This may have been due to the la ck of food the day of the experiment. In fact, Groff and Zinki (1999) reported a reduction in plasma glucose levels of fishes that were subjected to reduced feed or starvation. Consequently, to determine the effect of metomidate sedation on blood glucose levels as opposed to deficiency of data due to a lack of food, all koi were fed the day of the experiment for subsequent studies to satiation with Wardley TEN, TOTAL ESSENTIAL NUTRITION Pond Pellets. Koi Metomidate Concentration Range Finding Pilot Study A m etomidate concentration pilot study was conducted to determine the concentration required to induce a sedative stage of anesthesia as described by Stoskopf (1995) within five minutes of anesthetic exposure. Methods Experimental Design The koi were the sam e fish as previously de scribed on page 42, Experimental Design. There were 23 days between the previous experime nt and this experiment before this group of fish was used again. Koi were maintained in the same recirculating tank system previously described and water quality was analyzed as before (pages 42 Experimental Design).

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53 Water quality in the recirculating tank system was maintained at 0.3.5 mg/L total ammonia; unionized ammonia of 0.06.10 mg/L; 0.7 mg/L nitrite; pH of 8.2.5; temperature of 24.6.8C; alkalinity of 120 mg/L; hardness of 188 mg/L; carbon dioxide of 5 mg/L; and dissolved oxygen of 7.2.5 mg/L. The nitrite le vel in the system was above the potential toxic limit; however, chloride ions provided from salt (2.5g/L) addition to the system prevented the effects of hypoxia, if any. Although the unioni zed ammonia and nitrite levels were above the levels that are potentially toxic, the ko i did not display any signs of toxicity. A metomidate stock solution (page 50 Experimental Design) was used to dose 3.78 L aerated well water at a salinity of 3 g/L in a 7.57-L tank with an air stone. The solution was then mixed for 30 seconds. Because there were no stat istical differences in blood glucose levels in the previous study with metomidate concentr ations ranging from 0 to 1.0 mg/L, metomidate concentrations of 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0 mg/L were sequentially evaluated. The observation tank was emptied, rinsed, and refilled with untreated, aerated well water between each metomidate treatment observation. Observations For each m etomidate treatment, two koi were placed into the tank for observations of behavior and stage of an esthesia induced. Observations were recorded every minute for five minutes total. Koi were exposed to metomidate only once, and were pl aced back into their original tank on the recirculating tank system once observation time ceased. Results All m etomidate concentrations induced sedation within five minutes; however, narcosis was induced when koi were exposed to metomidate concentrations greater than 4.0 mg/L (Table 3-1).

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54 Discussion The m etomidate concentration required to i nduce a sedative stage of anesthesia within 5 minutes was 2.0 mg/L. Metomidate concentrati ons of 3.0 and 4.0 mg/L also sedated koi within 5 minutes. Koi 48 Hour in High Dose Metomidate Pilot Study This study determ ined the anesthetic stage induced following metomidate exposure over a 48 hour period in treatment under simulated trans port. After 48 hours, koi were placed back into their original tank on the recirculat ing tank system and recovery was observed. Methods Experimental Design The koi were the sam e fish as previously described on page 42, Experimental Design. There were 5 days between the previous experiment and this experiment. Koi were maintained in the same recirculating tank sy stem previously described and water quality was analyzed as before (pages 42 Experimental Design). A metomidate stock solution (page 50 Experi mental Design) was used to expose koi to simulated transportation in metomidate concentr ations of 1.0, 2.0, 3.0, and 4.0 mg/L. There were two koi per replicate and tw o replicates per treatment. Simulated Transportation The two boxes were placed side-by-side on a wood pallet and experienced sim ulated transportation as previously described (pag e 51 Simulated Transportation) (Figure 3-1). Observations Koi were observed for recovery after exposure to anesthesia post sim ulated transportation at 0 hour, 15 minutes, 2 hours, and 4 hours.

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55 Results After 48 hours in treatm ent all koi observed we re swimming upright in treatment bags in a sedative stage of anesthesia for all concentratio ns of metomidate (Table 3-2). After 15 minutes in recovery tanks, koi exposed to 3.0 and 4.0 mg /L metomidate were swimming tilted to one side or bouncing along the bottom of the tank in deep sedation. These koi required approximately 2 hours to return to normal behavior compared to koi exposed to lower concentrations of metomidate which required approximately 15 minutes to recover normal behavior. Discussion Koi exposed to m etomidate in concentr ations of 1.0, 2.0, 3.0, and 4.0 mg/L for 48 hours were sedated. The time required for transporte d fishes to resume normal behavior is an important consideration as customer perception of abnormal beha vior may potentially affect sales. There was a wide range of metomidate concentrations that induced sedation in koi. Koi Plasma Cortisol and Blood Glucose Pilo t Study A pilot study was conducted with koi to determine the effect of metomidate exposure on plasma cortisol and blood glucose levels following 24 hours in simulated transportation. Methods Experimental Design The koi were the sam e fish as previously described on page 42, Experimental Design. There were 4 days between the previous experiment and this experiment. Koi were maintained in the same recirculating tank sy stem previously described and water quality was analyzed as before (pages 42 Experimental Design). Water quality in the recirculating tank system was maintained at 0.3.6 mg/L total ammonia; unionized ammonia of 0.06.12 mg/L; 0.7 mg/L nitrite; pH of 8.2.6; temperature of 24.6.8C; alkalinity of 137 mg/L; ha rdness of 137 mg/L; carbon dioxide of 5

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56 mg/L; and dissolved oxygen of 7.1.7 mg/L. The nitrite level in the system was above the potential toxic limit; however, salt was maintained in the system at 2 g/L and provided chloride ions that prevented effects of hypoxia, if any. Although the unionized amm onia and nitrite levels were above the level at which toxicity may occu r, the koi did not display any physical signs of toxicity. In addition, adverse system water quality did not appear to affect blood glucose levels which were within the normal range for plas ma glucose in common carp of 30 mg/dL (Groff and Zinki 1999). Koi were exposed to simula ted transportation conditions for 24 hours in metomidate concentrations of 0.50, 0.75, 1.0, 2.0, 3.0, and 4.0 mg/L. There were two koi per replicate and two replicates per treatment. A metomidate stock solution was used (page 50 Experimental Design) to dose the water. Simulated Transportation Koi underwent sim ulated transportation as pr eviously described (page 51 Simulated Transportation). Blood Collection and Blood Chemistry Blood was collected and blood glucose level was analyzed as described previously (page 45 Blood Collection and Blood Che mistry). To analyze plasma cortisol, the plasma samples were diluted 400 times with EIA buffer for koi treated at metomidate concentrations of 3.0 and 4.0 mg/L. Plasma samples from koi treated at metomidate concentrations of 0.5, 0.75, 1.0, and 2.0 mg/L were diluted 600 times. The plasma cor tisol methods previously described were then followed (pages 46 Blood Collection and Blood Chemistry). Results All plasm a cortisol levels were within the e xpected range for the kit. However, there was not enough plasma for cortisol analysis in two blood samples, one from each of 0.75 and 1.0

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57 mg/L metomidate. Cortisol levels were si gnificantly higher for koi treated with 0.5 mg/L metomidate (251.8 ng/mL) compared to those treated with 1.0, 2.0, 3.0, and 4.0 mg/L metomidate (23.3.6 ng/mL) (ANOVA: F = 10.42; df = 5, 16; P < 0.0001) (Figure 3-5). Blood glucose levels of koi at metomidate concentrations of 0.5 mg/L (97.8 mg/dL) and 1.0 mg/L (84.0 mg/dL) were significantly highe r than in koi treated with metomidate concentrations of 3.0 mg/L (34.3 mg/dL) a nd 4.0 mg/L (37.5 mg/dL) metomidate (ANOVA: F = 5.90; df = 5, 18; P = 0.0021) (Figure 3-6). Discussion Plasm a cortisol levels in koi exposed to metomidate concen trations greater than 1.0 mg/L were within the range for unstressed fishes. Also, an increase in bl ood glucose levels was inhibited in koi treated at the higher metomidate concentrations, 2.0, 3.0, and 4.0 mg/L. Consequently, metomidate concen trations of 1.0, 2.0, 3.0, and 4.0 mg/L were utilized for the koi experiment. In addition, increasing the dilution factor for plasma samples from koi transported at lower concentrations of meto midate yielded plasma cortisol levels within the range of the assay. Therefore, the use of the increased dilution factor, 600 times, was effective for the analysis of plasma cortisol levels from koi transported at lower concentrations of metomidate. Koi Fright Substance (Schreckstoff) Pilot Study This study was conducted on koi to determ ine if trauma to the epidermis from netting and blood collection would induce a phy siological response in koi that were not subjected to handling. Plasma cortisol and whole blood glucose le vels were analyzed as indicators of stress. Methods Experimental Design The koi were the sam e fish as previously described on page 42. There were 3 months between the previous experiment and this experi ment. Twenty three koi were held in two 75.7-L

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58 tanks with 37.8 L of well water, an air stone, an d a sponge filter. One tank of 12 koi was located above another tank of 11 koi. The two tanks were separate from the recirculating tank system. The two tanks were connected to each other by water running from the top tank into the bottom tank via an overflow drain line. A pump in the bottom tank return ed water to the top tank. The flow rate of the pump in the bottom tank was calculated by observing the time required to fill a 1-L graduated cylinder. Based on the flow rate, the total volume of water in the top tank theoretically cycled th rough to the bottom tank in nine minut es, although it is un likely that 100% of the water in the top tank cy cled to the bottom tank. Both tanks were covered on the bottom and sides with black garbage bags, and an opaque polystyrene lid was used to cover the tops of the tanks to minimize visual stress. Koi resided in the connected ta nks for three days prior to the experiment. Blood Collection and Blood Chemistry The day of the experim ent, four koi were removed from the top tank for blood collection and then returned to the tank immediately after blood collection. Twelve minutes after koi from the top tank had been sampled four koi from th e bottom tank were sampled. The sampling time period allowed for one complete water exchange from the top tank into the bottom tank, and for plasma cortisol and blood glucose levels in the koi in the bottom ta nk to change, if at all. Blood collection methods previously described were followed (page 45 Blood Collection and Blood Chemistry). Blood glucose and plasma cortisol were analyzed with the methods previously mentioned (pages 45 Blood Collection and Blood Chemistry). Results Koi from the bottom tank had significantly hi gher plasma cortisol levels (50.5 ng/mL) than koi that were sampled from the top tank (26.7 ng/mL) (ANOVA: F = 10.51; df = 1, 6; P = 0.02) (Figure 3-7). However, there was no signifi cant difference in blood glucose levels for koi

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59 sampled first from the top tank (56.0 mg/dL) compar ed to koi sampled twelve minutes later from the bottom tank (39.0 mg/dL) (ANOVA: F = 4.59; df = 1, 6; P = 0.07) (Figure 3-8). Discussion The higher plasm a cortisol levels for koi th at were sampled from the bottom tank suggest that these koi had a physiological response to the sampli ng of the koi in the top tank. These koi may have reacted to Schreckstoff substance that was potentially released by the koi that were handled. The lack of significant difference in bl ood glucose levels may have been due to a lag in the production of blood glucose in response to the stressor. The potential for fright substance release pr ompted the addition of a granular activated carbon (GAC) filter to the sump at the return wate r line of the recirculating tank system for the duration of the koi experiment. The purpose of th e GAC filter was to aid in the adsorption of Schreckstoff substance that was released during handling and sampling of the koi. Pilot Studies Discussion Results f rom the pilot studies indicated that koi required metomidate concentrations at the upper end of the manufacturers recommended dose and higher (1.0.0 mg/L) to induce sedation within the chosen time frame of 5 minut es. However, the same group of koi was used throughout the pilot studies, and the effect of repeated metomi date exposure is not known. Smith et al. (1999) found that repeated exposure to tricaine methane sulfonate significantly reduced induction time after the third weekly expos ure in hybrids of whil e tilapia (Nile tilapia O. niloticus x blue tilapia O. aureus ) crossed with Mozambique tilapia O. massambicus The same group of koi was used for all pilot studies due to budgetary constraints. The increase in plasma cortisol and blood glucose levels after simula ted transportation illustrated the physiological effect of transportation stress. Due to the potential deleterious effects of this stressor, the

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60 investigation of the use of tran sportation sedatives that may redu ce or inhibit this physiological response was justified. The life support equipment of th e recirculating tank system did not have adequate time to establish sufficient numbers of nitrifying bacteria; as a result, nitrite and unionized ammonia were consistently at levels potentially toxic to fishes. However, chloride ions from the addition of salt were available to counteract the potential toxic e ffects of nitrite, if any. But, no signs of nitrite or unionized ammonia toxicity were obser ved in the koi at any time during the pilot studies. Water quality parameters in the recirculati ng tank system were tested once a week. Analysis of water quality parameters on a more frequent basis would have provided a better understanding of the fluctuations of the total ammonia and nitrite in the system. The additional information may not only have been useful in monitoring and adjusting water quality as necessary, but also reduce the potential for adverse effects to koi plas ma cortisol and blood glucose levels. Nonetheless, adverse system wate r quality did not appear to affect the koi blood glucose or plasma cortisol levels, both of which were within normal ranges for common carp (Tanck 2000, Goos and Conste n 2002, Palmeiro et al. 2007).

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61 Table 3-1. Observations of two koi placed into a 7.57-L tank with 3.78 L aerated well water. a Treatment (mg/L) 1 minute 2 minutes 3 minutes 4 minutes 5 minutes 2.0 Light sedation Light sedation Li ght sedation Light sedation Light sedation 3.0 Deep sedation Light sedation Li ght sedation Light sedation Light sedation 4.0 Light/deep sedation Light sedation Light sedation Light/deep sedation One fish light sedation, other fish light/deep sedation 5.0 Light sedation Light sedation Light sedation Deep sedation Light narcosis 6.0 Light sedation Light sedati on Light sedation Light/deep sedation Light narcosis 7.0 Light sedation Deep sedation Deep sedation Deep sedation/light narcosis One fish light narcosis, other fish deep narcosis 8.0 Light sedation Deep sedation One fish deep sedation, other fish deep narcosis One fish deep sedation, other fish deep narcosis Deep narcosis 9.0 Deep sedation One fish deep sedation, other fish light narcosis Deep narcosis Deep narcosis Deep narcosis 10.0 Light sedation Deep sedation Deep narcosis One fish deep narcosis, other fish light anesthesia Light anesthesia a Koi were observed for sedation level every mi nute for five total minutes. Metomidate concentrations were raised in crementally by 1 mg/L starting w ith 2.0 mg/L and ending at 10.0 mg/L. Koi were used only once with tank wate r replaced with each new concentration used.

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62 Table 3-2. Observations of koi stage of anesth esia after 48-hour simulated transportation with metomidate exposure and observati ons of recovery post-exposure. a Metomidate (mg/L) Time Observations 1.0 48 Hours in treatment Light sedation 1.0 48 Hours in treatment Light sedation 2.0 48 Hours in treatment Light sedation 2.0 48 Hours in treatment Light sedation 3.0 48 Hours in treatment Light sedation 3.0 48 Hours in treatment Light sedation 4.0 48 Hours in treatment Light/deep sedation 4.0 48 Hours in treatment Light/deep sedation 1.0 15 Minutes in recovery Normal behavior 1.0 15 Minutes in recovery Normal behavior 2.0 15 Minutes in recovery Normal behavior 2.0 15 Minutes in recovery One nor mal behavior, one Light sedation 3.0 15 Minutes in recovery Light sedation 3.0 15 Minutes in recovery Light/deep sedation 4.0 15 Minutes in recovery Deep sedation 4.0 15 Minutes in recovery Deep sedation 1.0 2 Hours in recovery Normal behavior 1.0 2 Hours in recovery Normal behavior 2.0 2 Hours in recovery Normal behavior 2.0 2 Hours in recovery Normal behavior 3.0 2 Hours in recovery Normal behavior 3.0 2 Hours in recovery Normal behavior 4.0 2 Hours in recovery Normal behavior 4.0 2 Hours in recovery Normal behavior 1.0 4.5 Hours in recovery Normal behavior 2.0 4.5 Hours in recovery Infrequent erratic body contractions 2.0 4.5 Hours in recovery Normal behavior 3.0 4.5 Hours in recovery Hiding at back of tank under stand pipe 3.0 4.5 Hours in recovery Normal behavior 4.0 4.5 Hours in recovery Normal behavior 4.0 4.5 Hours in recovery Normal behavior a There were two koi and two replicat es per metomidate concentration.

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63 Figure 3-1. Simulated transportation fish shi pping box arrangement. Each shipping box (432 mm x 432 mm) contained four square-bottom, quarter-size, plastic shipping bags (191 mm x 165 mm x 500 mm). The compressor on the pallet agitated the pallet for 30 seconds every 16 minutes and each air pump placed on the top of each stack of boxes agitated the boxes constantly for the duration of the experiment. Figure 3-2. Koi plasma cortisol levels from cortisol valida tion pilot study post simulated transportation. Letters denote si gnificantly different groupings ( P < 0.05) with standard error bars.

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64 Figure 3-3. Koi blood glucose levels from the cortisol validation pilot study post simulated transportation. Letters denote si gnificantly different groupings ( P < 0.05) with standard error bars. Figure 3-4. Koi blood glucose levels from th e blood glucose pilot study. The vertical line separates baseline koi from koi exposed to simulated transportation.

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65 Figure 3-5. Koi plasma cortisol levels from the plasma cortis ol and blood glucose pilot study after 24 hours in simulated transportation. Letters denote significantly different groupings ( P < 0.05) with standard error bars. Figure 3-6. Koi blood glucose levels ( N = 4 koi per treatment) from the plasma cortisol and blood glucose pilot study following 24 hours in simulated transportation. Letters denote significantly different groupings ( P < 0.5) with standard error bars.

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66 Figure 3-7. Koi plasma cortisol levels from Schreckstoff subs tance pilot study. Tanks were connected to each other with koi in top tank sampled first and koi in the bottom tank sampled 12 minutes later. Letters de note significantly different groupings ( P < 0.05) with standard error bars. Figure 3-8. Koi blood glucose levels from Schreckstoff substance pilot study. Tanks were connected to each other with koi in top tank sampled first and koi in bottom tank sampled 12 minutes later.

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67 CHAPTER 4 KOI EXPERIMENT Introduction Transportation of live ornam ental fishes from fa rm to market is stre ssful and may lead to physiological changes such as increased plas ma cortisol and glucose levels, decreased marketability because of loss of scales and fi n erosion, and mortality. For example, several studies have demonstrated significan t increases in plasma cortisol levels of fishes in response to handling (Pickering and Pottinger 1989, Avella et al. 1991), net confinement (Woodward and Strange 1987, Pickering and Pottinger 1989), lowered tank water volume (Davis and Griffin 2004), and stocking density (Barcellos et al. 1999). Cortisol is a steroid hormone synthesized by the interrenal tissue in teleost fishes (Haz on and Balment 1998, Norris and Hobbs 2006) and is commonly used as an indicator of stress (Al-Kindi et al. 2000). Even small increases in plasma cortisol levels may be harmful by adversely a ffecting immunity (Brown 1993). A significant elevation in plasma cortisol leve ls is frequently observed 5 mi nutes after the in itiation of an acute stressor (Bart on and Iwama 1991, Sumpter 1997, Ruan e 2001). Ilan and Yaron (1976) demonstrated that collecting blood by cardiac pu ncture within 4 minutes did not elicit an appreciable rise in plasma co rtisol levels in unanesthetized carp, sp ecies unknown. Plasma cortisol production stimulates glucose production precursors from peripheral stores and increases the livers capacity to produce glucose (Mommsen et al. 1999, Al-Kindi et al. 2000). Glucose level is also frequently used as an indicator of stress. The deleterious effects of transportation may be reduced by use of an anesthetic agent in shipping water. Sedation, a stage of anesthesia, may minimi ze the physiological response by lowering the fishes perception of a stressor (Stoskopf 1995). Sedation is achieved when evidenced by reduced movement and ventilatio n without interference with equilibrium, or

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68 osmoregulation (Stoskopf 1995). Oxygen cons umption and nitroge nous waste output are reduced during transport when fishes are sedated; thus sedation may furt her reduce stress associated with the degradation of shippi ng water quality in the transport container (Wedemeyer 1996a) However, the use of an anesthetic ag ent for transportation th at induces a stage of anesthesia deeper than sedation, such as narc osis, should be avoided because not only does it interfere with osmoregulation, but fishes may sink to the bottom of the shipping container. This may create localized areas of water quality de gradation such as lo w dissolved oxygen (DO) (Ross and Ross 1999). An important consideration th at may affect marketabi lity of fishes is the recovery time (i.e., return to nor mal state without behavioral side effects) following exposure to an anesthetic during transporta tion. Customer perception of a bnormal behavior may potentially affect the sales of fishes. There are a variety of anesthetic agents currently used in th e ornamental fish industry but none are approved by the U. S. Food and Drug Administration (FDA) for transportation of fishes. Two commercial products used are FDAapproved for temporary immobilization of all finfish: Finquel (Argent Laboratories, Redmond, Washington) and Tricaine-STM (Western Chemical, Inc., Ferndale, Washi ngton), both of which contain tricaine methane sulfonate (MS222). However, there are disadvantages to the use of MS-222. Th is anesthetic often induces an initial excitatory respons e that may result in increased plasma cortisol production (Davis et al. 1982, Robertson et al. 1987, Ross and Ross 1999). Also, MS-222 continues to accumulate in tissues over time, possibly resulting in over-sedation and ventilatory arrest (Barton and Peter 1982, Summerfelt and Smith 1990, Ross and Ross 1999). In addition, MS-222 can lower the pH of poorly buffered shipping water, and thus an addition of sodium bicarbonate is required in

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69 many cases (Summerfelt and Smith 1990, Noga 2000). Given these negative aspects, there is a need for research that evaluates other an esthetic agents for use in aquaculture. Metomidate hydrochloride (hereafter referred to as metomidate) is an anesthetic agent that has been used in fishes, birds, and ma mmals, including pigs and humans (Branson and Booth 1995). Metomidate is an imidazole-based, hypnotic drug that is eff ective for anesthesia but not analgesia (Branson and Booth 1995, Ross and Ross 1999, Treves-Brown 2000). A commercial metomidate formulation, AquacalmTM (Syndel Laboratories, Ltd., Qualicum Beach, British Columbia, Canada) is currently approved for use in Canada as an anesthetic for non-food fishes in the order Siluriformes (i.e., catfish) a nd in the families Cyprinidae (e.g., carp, goldfish), Poeciliidae (e.g., live-bearers), Cent rarchidae (e.g., sunfish, bass), Cichlidae (i.e., cichlids), and Pomacentridae (i.e., saltwater damselfish). Metomidate appears to have advantages compared to MS-222. For example, several studies have demonstrated that metomidate reduces the elevation of plasma cortisol levels in stressed fishes such as threespot gourami Trichogaster trichopterus (Crosby et al. 2006c), fathead minnow Pimephales promelas (Palic et al. 2006), channel catfish Ictalurus punctatus (Small 2003), Atlantic salmon Salmo salar (Olsen et al. 1995, Ivers on et al. 1998, Iverson et al. 2003), juvenile and adult Chinook salmon Oncorhynchus tshawytscha (Kreiberg and Powell 1991, Sharpe 1998), juvenile striped bass Morone saxatilis (Bills et al. 1993), sunshine bass ( M. chrysops x M. saxatilis) (Davis and Griffin 2004), and red drum Sciaenops ocellatus (Robertson et al. 1988, Thomas and Robertson 1991). Metomi date is thought to reduce cortisol production by suppressing 11hydroxylation of cholestero l (Wada et al. 1988, Olsen et al. 1995). Therefore, the use of metomidate may reduce the physiological stress response in fishes as measured by plasma cortisol and blood glucose levels. In addition to suppressing cortisol

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70 production, metomidate has few adverse side e ffects (Wedemeyer 1996). Metomidate does not accumulate with long exposure time and has litt le effect on water chemistry (R. Bradshaw, Syndel Laboratories Ltd., pers onal communication). For exampl e, Guo et al. (1995a) reported that 1.0 mg/L metomidate had no effect on water pH, total ammonia, or carbon dioxide during 48 hours simulated transportation of southern platyfish Xiphophorus maculatus. Additionally, Guo et al. (1995b) found that oxygen cons umption was decreased in southern platyfish exposed to 1.0 mg/L metomidate. There is little published information available on metomidate use with ornamental fishes, and results of its use are varied and sometimes negative. Crosby et al. (2006c) reported significantly lower plasma cortis ol levels in threespot gourami exposed to 0.8 mg/L metomidate following acute handling stress compared to untrea ted control individuals. Metomidate was used by Hill et al. (2005) to an esthetize rainbow sharks Epalzeorhynchos frenatum prior to topical gill application of a spawning hormone; however, some of the rainbow sharks would intermittently react strongly to external stimuli (J. E. Hill, University of Fl orida, personal communication). Additionally, Massee et al. (1995) concluded that metomidate at 6 mg/L was not an effective anesthetic for larval goldfish Carassius auratus because of long induction and recovery time. Guo et al. (1995a) also found that southern pl atyfish exposed to 1.0 mg/L metomidate for 48 hours had 11% mortality compared to 6% in untr eated control individuals. Also, 5 days after exposure metomidate treated southern platyfish ha d 42% mortality compared to 12% in untreated control individuals (Guo et al. 1995a). Additionally, oxygen consum ption in southern platyfish exposed to metomidate increased as temperat ure increased from 20C to 30C compared to untreated control indivi duals (Guo et al. 1995b).

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71 The present study investigated the use of metomidate seda tion in koi transported for approximately 24 hours. Koi, an or namental variety of the common carp Cyprinus carpio is produced in Florida (Hill and Ya nong 2006) and several other states and shipped to wholesalers, distributors, retail stor es, and hobbyists around the world. Koi cu ltured in Florida represent 9% (i.e., $589,000) of the total koi fa rm-gate value in the U.S. (U SDA NASS 2006). For this reason they were chosen as the experimental model due to their eco nomic importance. The specific objectives were 1) to measure plasma cortisol and blood glucos e levels as indicators of physiological stress following transportation of koi in different concentrations of metomidate and 2) to determine the effect of metomidate se dation on marketability of transported koi as evaluated by visual assessment of appearance, behavior, and ac tivity level posttransportation. The objectives were tested through two separate experimental components: blood chemistry and marketability. Methods Experimental Design Koi ranging in size from 127 to 152 mm TL ( N = 300) were obtained from a Florida fish farm. Five koi were placed into each of 30, 75.7-L tanks for a total of 150 koi for each component of the experiment. The koi were held for two weeks prior to experimentation in a recirculating tank system that consisted of a su mp, a bubble-washed bead filter, an ultraviolet light sterilizer, a granular activated carbon filt er, and a fluidized bed filter that contained sand media. Each tank had an air stone, contained 37.8 L filtered well water, and drained into a common sump. Polystyrene partitions were placed between tanks to reduce the potential effect of visual stimuli from koi in ad joining tanks, and the tanks were covered to prevent the koi from jumping out. The photoperiod was 12 h light: 12 h dark with the excepti on of the day of the experiment (during data collectio n); the lights were on in the labor atory for a total of 36 hours to

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72 accommodate blood collection and observations. All koi were fed once daily to satiation, including the day of the experiment (W ardley TEN TOTAL E SSENTIAL NUTRITION Pond Pellets floating pellet, Hartz Mountain Corporation, Secaucus, New Jersey). Dissolved oxygen (DO), pH, and temperature in the recirculati ng tank system were measured weekly with an LDO digital mete r (HQ20, Hach Company, Loveland, Colorado). Immediately afterwards, total ammonia (NH3 + NH4 +), nitrite (NO2 -), alkalinity, hardness, and carbon dioxide (CO2) were analyzed with a Hach Freshw ater Fish Farmers Kit (FF-1A, Hach Company, Loveland, Colorado) and salinity was determined with a salinity refractometer (Aquatic Eco-Systems, Inc., Apopka, Florida). Wa ter quality in the recirculating tank system during the experiment ranged from 0.8 mg/L total ammonia; 0.16 mg/L unionized ammonia; 0.03.79 mg/L nitrite; pH 7.6.6; 22.8.8C temperature; 137 mg/L alkalinity; 171 mg/L hardness; 5 mg/L carbon dioxide; 7.3.1 mg/L dissolved oxygen, and 0 g/L salinity (NaCl Mort on Salt, Chicago, Illinois). Koi were exposed to one of five meto midate concentrations: 0, 1.0, 2.0, 3.0, and 4.0 mg/L. There were 5 replicate tanks with 5 koi for each treatment. In addition to the metomidate treatments, there were 5 baseline tanks of koi that was not transported or e xposed to metomidate. Experimental groups were randomly assigned to each tank. Three of the metomidate concentrations used in the experiment were high er than the labeled dosag e rate for cyprinids of 0.0 mg/L; however, these hi gher concentrations induced sedati on in pilot studies with similar sized koi (Chapter 3). For the blood chemistry com ponent of the experiment, the five koi in each tank were uniquely fin-clipped. Shipping water was dosed with a metomidate stock solution of 1 mg metomidate per 1 mL distilled water. Salt (NaCl) is typically added at 3 g/L for transportation of koi from

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73 Florida (J. Pawlak, Blackwater Creek Koi Farms, Inc., personal communication; R. Slay, Florida Fish Farms, Inc., personal communication). Therefore, salt was added to the shipping water at 4 g/L. Each bag with shipping water, salt, and metomidate was then mixed for 30 seconds. One and a half liters of shipping water was added to e ach of five square-bottom, quarter-size, plastic shipping bags (191 mm x 165 mm x 500 mm) per treatment. In Florida, shipping densities of 20 koi that range in size from 127 to 152 mm TL are transported per 7.57 L of shipping water in a square-bottom, full-size, pl astic shipping bag (393.7 x 368.3 x 571.5 mm) for up to 24 hours (C. V. Martinez, University of Florida, persona l communication; R. Sla y, Florida Fish Farms, Inc., personal communication); for these experiment s there were five koi per shipping bag with 1.5 L water. Each bag was filled with oxygen gas, clos ed with a rubber band, placed into a polystyrene, square, single shipping box ( 432 mm x 432 mm) with a lid, and each box was placed inside an outer cardboard box for labeling. Bags were randomly assigned to a box; there were four bags per box, and empty air filled bags were used as place holders as needed. In the blood chemistry component of the experiment, ba gging was staggered six minutes between each tank to allow time for blood collection and to standardize the sampling time. For the marketability component of the experiment, the bags were randomly assigned to a box, and the koi were bagged and set into the box es with no delay in bagging time. Transportation Boxes were transported via truck for approxi m ately 2.5 hours from Gainesville, Florida, to a distribution facility of ornamental fishes in Gibsonton, Florida. The boxes of koi were transported about 12 hours later by truck to Tamp a International Airport in Tampa, Florida and then flown via a domestic airline to North Carolin a. The boxes were then returned via domestic airline to the Gainesville Regional Airport in Gainesville, Florida, that same afternoon and

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74 driven via truck back to the res earch laboratory at the University of Florida, Program in Fisheries and Aquatic Sciences. The koi were in transport for approximately 24 hours. Blood Collection and Blood Chemistry Koi were sampled at 0, 2, 6, and 12 hours post-tr ansportation to determ ine the effect of metomidate exposure over time. One koi per tank was sampled at each sample time and each koi was identified by fin-clip and used only once. For blood collection, koi were placed in dorsal recumbency on a wetted foam pad and covered with a wet chamois cloth to restrain the animal during sampling. Blood samples of 0.5 mL were collected from the caudal vessels using a 25-G needle fitted on a 1-mL syringe that contained about 0.06 mL sodium hepa rin (Baxter Healthcare Corporation, Deerfield, Illinois). At time 0 hour post-transportation a bag was opened, and a koi was removed and immediately sampled. Remaining koi in the ba g were placed into th e original tank on the recirculating tank system for subsequent sampling. Plasma cortisol is frequently observed to increase 5 minutes after the initiation of a stressor (Barton and Iwama 1991, Tanck 2000, Ruane 2001); therefore, blood was collected within 4 minutes from when a net first entered a tank or bag. If 0.5 mL total volume blood and heparin was collected before the 4 minutes expired, blood glucose was imme diately analyzed. If 0.1.5 mL total volume was collected within 4 minutes, blood glucose was analyzed from the collection attempt. However, if less than 0.1 mL total volume was collected when the four minutes expired, sampling ceased and the koi was placed back into the original tank on the recirculating tank system. Immediately after blood was collected, blood glucose levels were analyzed with a handheld Ascensia ContourTM glucose meter (Bayer Healthcare, Morristown, New Jersey). The meter required 0.6 L of blood, analyzed a sample in 15 seconds, and had a detectable range of 10 mg/dL. Blood glucose levels were correc ted for heparin dilution. For blood glucose

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75 levels that were below the minimum level of de tection for the meter (i.e., less than 10 mg/dL) a value of 5 mg/dL was arbitrarily assigned. This allowed for the determination of significance between blood glucose levels for koi transported at the various concentrations of metomidate. After glucose analysis, any remaining blood in the syringe was transferred into a 1.5 mL microcentrifuge tube and held in a refrigerator at 4C for up to 2 hours prior to centrifugation. Whole blood was centrifuged (HN S-II, Dam on/IEC Divison, Needham, Massachusetts ) for 5 minutes at 2500 rpm and plasma was pipetted in to 1.5 mL microcentrifug e tubes. Collected plasma was placed into a -80C freezer for two m onths until analysis. Plasma samples may be frozen at or below -75C for up to 6 months w ithout significant change in composition (Houston 1990). Plasma cortisol levels were determined using a competitive plasma cortisol enzyme immunoassay (EIA) with a detectable range of 7.9 pg/mL (Cayman Chemical Company, Ann Arbor, Michigan). In preparation for analysis, plasma samples were thawed on ice. Ethyl ether was used to extract the lipophilic components from the hydrophilic components of the plasma. Extraction consisted of adding 5 mL ethyl ether to each 50 L plasma sample. The solution then sat undisturbed for one minute followe d by two minutes of vigorous vortex mixing (Vortexer, VWR LabShop, Batavia, Illinois). The samples were then left undisturbed for one minute, and the solution was placed into a dry ice-chilled methanol bath at less than or equal to -34.4C for two minutes. The ethyl ether containing the li pophilic components for each sample was then decanted into a clean test tube, and the ether wa s allowed to evaporate. The process was then repeated in the original test tubes with the pellet of hydrophilic components. The air dried tubes were then rehydrated a nd diluted with EIA buffer (Cayman Chemical Company, Ann Arbor, Michigan). Plasma samples from baseline koi were diluted 400 times and

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76 plasma samples from transported koi were diluted 600 times. Afte r sample dilution, the manufacturers methods were then followed. In brief, each diluted plasma sample was pipetted into wells on a 96-well assay plate (Cayman Chemi cal Company, Ann Arbor, Michigan) along with eight cortisol standards and a series of plate control samples. Each sample was run in duplicate. The cortisol standards and plate contro l samples were used to generate the standard curve for the plate. Once all samples were pipette d on the plate, it was inc ubated in a refrigerator at 4C for no more than 18 hours. After incubation, developing reagent (Cayman Chemical Company, Ann Arbor, Michigan) was added to each well. The developing reagent competed with the cortisol in the koi plasma samples to bind to a limited number of cortisol-specific binding sites on the plate. After addition of the developing reagent, the plate was then placed on an orbital shak er (Orbitron IITM, Boekel, Feasterville, Illinois) and allowed to develop at room temperature for 90 minutes. Samp le plates were read with a microplate spectrophotometer (Bio-Rad Benchmark Plus, Bio-Rad Technologies, In c., Hercules, California) at a wavelength of 412 nm. Plasma cortisol levels were determ ined using the Cayman Chemical Company (Ann Arbor, Michigan) cortisol analysis tool. The plasma cortisol le vels were then corrected for heparin dilution. There were 27 plasma cortisol samples outside the detectable range of the assay. Two months after the initial cortisol analysis an additional cortisol assay was completed with these 27 plasma samples following the same methodology at dilutions of 800 and 1000 times. The plate reader previously used was unavailable. Cons equently, the additional plate was read using a multimode plate reader (Synergy HT, Bio-Tek In struments, Inc., Winooski, Vermont) at the same wavelength (412 nm) used previously. On ce both plate readers were available for use, another cortisol assay was performed with 35 prev iously frozen samples, and the plate was read

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77 on both plate readers to determine if there wa s a significant difference between the two. A paired students t -test (Microsoft Office Excel 2003, Re dmond, Washington) performed on the data revealed that results from the two pl ate readers were not si gnificantly different ( t = 3.02, df = 34, P = 0.998); therefore, the plasma cortisol da ta obtained from both plate readers were combined for plasma cortisol analysis. Differences in mean blood glucose and plasma cortisol levels were tested with one-way analysis of variance (ANOVA) with a Type 1 error rate ( ) of 0.05 (JMP 5.1 Statistical Discovery Software, SAS Instit ute, Cary, North Carolina). Significant ANOVA were followed by Tukeys HSD comparison of means (JMP 5.1 Sta tistical Discovery Software, SAS Institute, Cary, North Carolina). Appearance, Behavior, and Ac tivity Level Observations The appearance, behavior, and activity level of koi in each tank were evaluated by four judges that used a ranked, categorical value syst em (Table 4-1). The koi were observed at six tim es post-transportation: 0, 1, 4, 8, 12 hours, and 7 days. Appearance was judged using a pre-determined scale of visual ch aracteristics including coloration, scale loss, ragged fins, hemorrhaging, and exophthalmia. Scores of 1 or 2 denoted unsellable fish, and scores of 3 to 5 were considered to be sellable fish. Behavior was judged for characteristics such as hiding, isolation from tank mates, hanging in the water column, piping, gasping, spinning, flashing, and loss of buoyancy. Sc ores ranged from 1 to 2 that indicated unsellable fish to scores of 3 to 4 that signified sellable fish. Activity was assigned as not active, active, and very active. Activity level scores ranged from 1 that designated not active to a score of 5 that denoted very active. Appearance, behavior, and activ ity level observation data sc ores were organized by rank sum. A Kruskal-Wallis (K-W) test was performed at = 0.05 in JMP 5.1 Statistical Discovery

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78 Software (SAS Institute, Cary, North Carolina) to determine differences in the mean ranks of observation scores. If there we re significant differences the data were further analyzed by Dunns comparison of means to determine diff erences among treatments (Hollander and Wolfe 1973). Due to the conservative nature of the Dunns comparison of means test, = 0.15 was used (Hollander and Wolfe 1973). In addition, the significance between proportions of tanks that had sellable koi to tanks that had unsellable koi was evaluate d using one-way analysis of variance (ANOVA) with a Type I error rate ( ) of 0.5 followed by Tukeys HSD comparison of means in JMP 5.1 Statistical Discovery Software (SAS Institute, Cary, North Carolina). Data were arcsine square-root transformed in orde r to meet normality assumptions of the ANOVA (Microsoft Office Excel 2003). Shipping Water Physicochemistry Dissolved oxygen, pH, and tem perature were m easured directly from shipping bags with the LDO digital meter. In addition a shipping water sample was collected from each bag and stored in a refrigerator at 4 C for approximately 20 hours. To tal ammonia and carbon dioxide were then determined with a Hach Freshwater Fish Farmers Kit. Differences in mean shipping water physicoche mistry parameters were tested with oneway analysis of variance (ANOVA) with a Type 1 error rate ( ) of 0.05 (JMP 5.1 Statistical Discovery Software, SAS Instit ute, Cary, North Carolina). Significant ANOVA were followed by Tukeys HSD comparison of means (JMP 5.1 Sta tistical Discovery Software, SAS Institute, Cary, North Carolina). Results Blood Chemistry At tim e 0 hour post-transportati on, plasma cortisol levels from koi transported with 0 mg/L metomidate (310.8 ng/mL) were 2.2 to 2.5 times higher than those from koi transported at

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79 3.0 mg/L metomidate (125.0 ng/mL) or 4.0 mg/L metomidate (139.3 ng/mL) (ANOVA: F = 10.75; df = 5, 19; P <0.0001) (Figure 4-1). Also at time 0 hour post-transportation the plasma cortisol levels from koi transported at 3.0 a nd 4.0 mg/L metomidate were not significantly different from plasma cortisol levels from baseline koi (34.8 ng/mL). At time 2 hours posttransportation blood glucose levels from koi tran sported with 1.0 and 4.0 mg/L metomidate were significantly higher than baseline fish (ANOVA: F = 4.10; df = 5, 23; P = 0.008) (Figure 4-2). Appearance, Behavior, and Ac tivity Level Observations At tim e 0 hour post-transportati on, there were no differences in appearance scores among all transported koi ( K-W : H = 12.54; df = 5, 24; P = 0.03). However, the appearance of koi transported at 1.0, 2.0, and 3.0 mg/L metomidate we re not significantly different from baseline koi whereas the koi transported with 0 and 4.0 mg/L metomidate had significantly lower scores (Figure 4-3). There was no difference in the pe rcentage of sellable tanks of koi based on appearance (Figure 4-4). There was no difference in koi behavior at any observation time (Figure 4-5) or the percentage of sellable tanks of koi based on be havior (Figure 4-6). At time 0 hour post-transportation koi transported with 4.0 mg/L metomidate were significantly more active than baseline koi; but, activity level of all transported koi were not signifi cantly different among metomidate concentrations ( K-W : H = 14.24; df = 5, 24; P = 0.01) (Figure 4-7). Shipping Water Physicochemistry For the blood chem istry component of the e xperiment there were no differences in the shipping water physicochemistry parameters, dissolved oxygen, total ammonia, carbon dioxide, or pH, analyzed at 0 hour post-tr ansportation (Table 4-2). Ho wever, koi transported with 0 mg/L metomidate had significantly higher shippi ng water temperature (23.3C) compared to koi transported at 1.0 mg/L metomidate (22.6C) and 3.0 mg/L metomidate (22.9C) (ANOVA: F = 11.75; df = 4, 20; P = <0.0001). Additionally, koi trans ported with 2.0 mg/L metomidate

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80 (23.1C) and 4.0 mg/L metomidate (23.0C) had hi gher temperatures than 1.0 mg/L metomidate. For the marketability component of the experiment there were no significant differences in total ammonia, carbon dioxide, pH, or temperature an alyzed at 0 hour post-transportation among metomidate concentrations (Table 4-2). However, dissolved oxygen was almost twice as high for koi transported with 2.0 mg/L metomidate (1 4.5 mg/L) compared to koi transported with 0 mg/L metomidate (7.5 mg/L) (ANOVA: F = 2.84; df = 4, 20; P = 0.05). Discussion Koi is an im portant commercial aquaculture species, and their marketability may be affected by transportation stress. However, the use of an anesthetic may lower their perception to this stressor. Metomidate anesthesia appears to have physiological advantages such as inhibition of elevated plasma co rtisol and glucose levels; but, as far as the author knows, this study reports the only information a bout its use for transporting koi. In this study, koi were transported for a pproximately 24 hours, a typical commercial transport time. Metomidate con centrations of 3.0 and 4.0 mg/L in shipping water inhibited an increase in koi plasma cortisol levels; however, the use of metomidate during transportation did not provide prolonged inhi bition of plasma cortisol increas e once the koi were removed from metomidate exposure and placed into recovery tanks In comparison to baseline koi at time 0 hour post-transportation, plasma cortisol levels of koi transported with 0 mg/L metomidate were elevated indicating that the transportation event wa s stressful. As expected, the range of plasma cortisol levels from baseline koi at samp ling time 0 hour (7.5.2 ng/mL) were similar to reported baseline levels for the species ( 5 ng/mL) (Tanck 2000, Goos and Consten 2002). The increase in plasma cortisol levels at time 2 hour post-transpor tation in baseline koi that were not transported may have been due to th e visual stress of repeated movement in and out of the room at different sampling times. The ro om lights were on continuously the day of the

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81 experiment, and disruption of the normal 12 h ligh t: 12 h dark photoperiod may have affected koi plasma cortisol levels. There is no eviden ce that suggests that phot operiod affects plasma cortisol levels in koi; however, increasing plasma cortisol with increasing photoperiod has been observed in other fishes. Pavlidis et al. (1999) repor ted that the porgy Dentex dentex had significantly higher plasma cortisol levels that resulted from an increase in the photoperiod from 12 h light: 12 h dark to 16 h light: 8 h dark. Similarly, Almazon-Rueda et al. (2005) demonstrated higher plasma cortisol levels afte r an increase in the photoperiod from 6 h light: 18 h dark to 18 h light: 6 h da rk in the African catfish Clarias gariepinus The lack of differences in blood glucose leve ls between metomidate concentrations at sample time 0 hour post-transportation was une xpected because plasma cortisol production stimulates glucose production (Woodward a nd Strange 1987, Wedemeyer 1996a, Schreck et al. 1997). Therefore, a trend similar to that observed in the plasma cortisol levels was anticipated in the blood glucose levels. For example in a study by Baker et al. (2005), th e plasma cortisol and blood glucose levels of Atlantic sturgeon Acipenser oxyrinchus and shortnose sturgeon Acipenser brevirostrum increased in response to hypoxia stress. Additionally, in Atlantic salmon Salmo salar exposure to air for 15 seconds resulted in elevated plasma cortisol and glucose levels compared to control fish (Fast et al. 2008). Similar to the present study, Tanck et al. (2000) found that common carp stressed by cold shock had an increase in plasma cortisol levels but no change in plasma glucose levels compared to untreated control individuals. Comparable results have also been re ported in other fish species such as the brown trout Salmo trutta exposed to fluctuating water flow (Flodmark et al. 2002 ) and in an anesthetic efficacy study with the Atlantic salmon (Iversen et al. 2003). As expected, the range of blood glucose levels from baseline koi, 19.0.6 ng/mL, at sampling time 0 hour post-transportation were consistent with

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82 unstressed common carp plasma glucose levels previously reported (2026 ng/mL) (Pottinger 1998, Groff and Zinki 1999, Palmeiro et al. 2007). A common practice is to w ithhold feed from the fishes for 1 days prior to transportation to allow the digestive tract to be purged; this practice aids in maintenance of shipping water quality (Wedemeyer 1996a, Ro ss and Ross 1999, Lim et al. 2003). In pilot studies with koi, the glucose meter was not sensitive enough to register the blood glucose levels of several koi starved for one day prior to simu lated transportation. Wedemeyer et al. (1990) and Groff and Zinki (1999) repor ted lower plasma glucose levels in fishes that are fed less than normal or starved compared to fed fishes. Theref ore, the koi were fed the day of the experiment, and almost all of the blood glucose levels obta ined were high enough to register on the glucose meter. Similarly, Olsen et al (2008) found that Atlantic cod Gadus morhua fed prior to transport had higher plasma glucose levels compared to control fish one hour after acute stress. The appearance of koi transported with 1.0, 2.0, and 3.0 mg/L metomidate were not different from baseline koi that were not tran sported, and these concentrations may be a good choice to prevent deterioration of koi appearance during tran sportation. Although there was a potential beneficial e ffect on the appearance of koi expos ed to1.0, 2.0 and 3.0 mg/L metomidate, this effect was not observed in koi behavior or activity level. The positive effect on koi appearance may have been due to reduced activit y during transportation, and therefore resulted in less physical trauma. However, koi activity during transport was unknown. Transportation had a negative impact on koi appearance with 5% of all transported tanks of koi unsellable based on appearance during the initial observation after transportation. Th e plasma cortisol and appearance results at time 0 hour post-transportation indicate the be st metomidate concentration for transportation of koi is 3.0 mg/L.

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83 Metomidate had little effect on shipping water physicochemistry parameters. These results were similar to those reported by Guo et al. (1995a) on the effect of metomidate on total ammonia, carbon dioxide and pH in tran sportation of the southern platyfish X. maculatus However, these results contradict Wedemeyers ( 1996a) statement that the use of sedation aids in reducing oxygen consumption and total ammonia and carbon dioxide production in transport water. Although there was a stat istical difference in the wate r temperature among metomidate treatments, the difference was less than 1C a nd there was no difference in dissolved oxygen concentrations, the water physicochemistry pa rameter most affected by temperature. Additionally, the plasma cortisol levels in co ntrol koi were consider ably higher than koi transported with 3.0 and 4.0 mg/L metomidate although there was no difference in temperature among these treatments. Therefore, the temperat ure differences likely had no effect on the physiological response of the koi to transportation. In the ma rketability component of the experiment, dissolved oxygen was almost twice as high in shipping water treated with 2.0 mg/L as compared to the level in koi transported with no metomidate. This is unlike the results from the blood chemistry component and supports Wede meyers statement that sedation minimizes oxygen consumption. This suggests a beneficial effect of metomi date use for maintenance of dissolved oxygen levels during trans port at this concentration. This also suggests that the use of 2.0 mg/L metomidate may be useful for transportation of short duration of koi with ambient air filled bags as opposed to oxygen gas filled bags. However, because these results were not duplicated in the blood chemistry component of th e experiment, additional studies are needed to clarify the effect of metomidate, if any, on dissolved oxygen consumption. Water quality in the recirculating system throughout the experiment had nitrite levels greater than 0.10 mg/L and unioni zed ammonia levels greater than 0.05 mg/L and were above

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84 the levels considered potentia lly toxic to the koi (Wedemeyer 1996a). Chloride ions were available from salt addition to reduce the effect, if any, of nitrite toxicity; however, the salt concentration in the recirculating tank system fl uctuated between 0 and 3.0 g/L. Nonetheless, there were no behavioral signs of nitrite toxicity (e.g., leth argy and crowding near aeration equipment or water outlets) or ammonia toxi city (e.g., hyperventilati on and hyperexcitability) (Wedemeyer 1996a) observed in the koi at any time and these parameters likely did not alter the outcome of the experiment. An interesting note in this experiment was th e observation that koi transported at 3.0 and 4.0 mg/L metomidate appeared to be in light narcosis when the boxes were opened. However, the behavior and activity data from this experime nt do not reflect this obs ervation. In contrast, pilot studies with similar si zed koi exposed to 3.0 and 4.0 mg/L metomidate in simulated transport for 48 hours were observed to be sedated in the shipping ba gs during 24 and 48 hours of metomidate exposure. The difference in anes thetic stage observed may have been due to multiple metomidate exposure in pilot study koi whereas the experimental koi in this study were only exposed to metomidate once. There is cu rrently no data on repeated metomidate exposure in koi; however, Smith et al. (1999) reported that repeated exposure to tricaine methane sulfonate significantly reduced induction time after the third weekly ex posure in tilapia hybrids (Nile tilapia O. niloticus x blue tilapia O. aureus ) crossed with Mozambique tilapia O. mossambicus Whether the difference in anesthetic stage observe d was due to repeated metomidate exposure is not known at this time. The choice of anesthetic for transportation of koi may be determined by its biological effects on both fishes and humans and the cost of an effective dose. Tricaine methane sulfonate (i.e., MS-222) is a widely used anesthetic for te mporary immobilization of finfish (United States

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85 Food and Drug Administration); however, MS-222 may cause increased plasma cortisol production (Barton and Peter 1982, Da vis et al. 1982, Robertson et al. 1987). In addition, in humans, tricaine methane sulfonate dust may irritate lungs thr ough inhalation or skin through absorption (Ross and Ross 1999), and chronic expos ure is retinotoxic (Ber nstein et al. 1997). Unlike MS-222, metomidate exposure has a reported inhibitory effect on plasma cortisol increase in fishes (Robertson et al. 1988, Th omas and Robertson 1991, Iverson et al. 2003). Although metomidate use does not have the deleterious biological effects that are associated with MS-222, the cost of anesthetizing koi for trans portation at a metomidate dosage rate, 3.0 and 4.0 mg/L, which results in inhibition of plasma cor tisol increase, may be a limiting factor. At the current cost of metomidate, 4 treatments would average one dollar. Compared to number of treatments, depending on dosage rate, one dollar would buy 48 treatments of clove oil, 40 478 treatments of Aqui-STM, 38 treatments of MS-222, and 210,000 treatments of quinaldine sulfate. Ultimately the choice of anesthetic used is dependent on preference. A metomidate concentration of 3.0 mg/L durin g transport resulted in lower koi plasma cortisol levels and an increase in overall app earance. In addition, a non-significant, inverse relationship between the metomidate concentration and the plasma cortisol levels was observed. As the metomidate concentration increased, the pl asma cortisol level decreased. An important consideration is that lowered pl asma cortisol and blood glucose le vels do not necessarily signify that the koi are not stressed; but ra ther that the ability of the koi to respond to transport stress is reduced (Alistair Webb, University of Florida, personal communication). Further studies with koi that include additional biological indices of stress su ch as blood osmolality, blood hematocrit, and blood leucocrit shou ld be investigated. This init ial investigation suggests that

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86 there are benefits to transportati on of koi with metomidate. But more research is required to fully understand the implications of metomidate use with this species.

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87 Table 4-1. Criteria for evalua tion of koi marketability base d on appearance, behavior, and activity level observations post-transportation. Score Criteria Considerations Appearance 1 Very Bad-Unsellable More than 50% of koi abnormal with minor to moderate damage of eyes/body/fins Coloration, hemorrhaging, scale-loss, deformities, ragged/torn fins, clamped fins, exophthalmia, cloudy eyes, lesions, 2 Bad-Unsellable % of koi abnormal with minor to moderate damage of eyes/body/fins parasites 3 Average Less than or equal to 25% of koi abnormal, eyes/body/fins damage minor 4 Good Koi overall look normal, very little eyes/body/fins abnormalities 5 Excellent Koi overall extremely healthy looking, vibrant color, eyes/body/fins superior Behavior 1 Very Bad-Unsellable More than 50% of koi abnormal Piping, gasping, spinning, flashing, loss of buoyancy control, isolated/hiding, located in abnormal part of water column 2 Bad-Unsellable % of koi abnormal 3 Average Less than or equal to 25% of koi abnormal 4 Excellent All koi behaving alert Activity Level 1 Not Active Overall activity 2 Less Active 3 Active 4 More Active 5 Very Active

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88Table 4-2. Mean (SD) koi shipping water physicochemistry parameters. Different letters indicate significan t differences acros s rows ( P < 0.05). Experiment Metomidate (mg/L) Component Parameter a 0 1.0 2.0 3.0 4.0 F-ratio b Pr > F Blood Chemistry DO 8.93.7 12.93.2 13.03.1 10.72.5 11.82.5 1.61 0.21 TA 38.41.7 37.62.3 37.02.2 36.84.6 34.05.3 1.11 0.38 CO2 13221.2 12730.1 11723.3 12021.2 11527.4 0.40 0.81 pH 6.60.1 6.70.1 6.70.1 6.70.0 6.60.0 0.68 0.61 Temp C 23.30.1 z 22.60.3 x 23.10.2 yz 22.9.6 xy 23.0.1 yz 11.75 <.0001 Marketability DO 7.53.3 z 11.03.3 yz 14.53.9 y 13.54.8 yz 10.91.9 yz 2.84 0.05 T A 19.22.4 22.83.8 22.46.1 16.22.2 17.02.2 3.44 0.74 CO2 844.7 853.2 757.7 7522.1 7813.5 0.34 0.85 pH 6.60.1 6.70.1 6.70.1 6.70.0 6.70.0 0.68 0.61 Temp C 24.90.8 25.00.5 24.90.7 25.00.8 25.11.1 0.07 0.99 a DO, TA, and CO2 are in mg/L; Temp C = temperature in degrees Celsius. b Numerator df = 4, denominator df = 20.

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89 Figure 4-1. Mean plasma cortisol levels of tr ansported koi sampled at times 0, 2, 6, and 12 hours post-transportation. Letters denote significantly different groupings ( P < 0.05) with standard error bars. The vertical line separates base line koi from transported koi.

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90 Figure 4-2. Mean blood glucose levels of transporte d koi sampled at times 0, 2, 6, and 12 hours post-transportation. The vert ical line separates baselin e koi from transported koi.

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91 Figure 4-3. Koi appearance scores ( N = 25 koi observed for each metomidate treatment per observation time) from time 0 to 12 hours a nd 7 days post-transportation represented as a percentage of occurrence. Categorical value scale; 1 = very bad, >50% abnormal, 2 = bad, >25% abnormal, 3 = average, 25% abnormal, 4 = good, <2% abnormal, and 5 = excellent, 100% normal. Letters denote significantly different groupings ( P < 0.05). The vertical line separates baseline koi from transported koi.

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92 Figure 4-4. Koi appearance as a percentage of o ccurrence of sellable versus unsellable tanks of fish ( N = 25 koi observed for each treatment pe r observation time) from time 0 to 12 hours and 7 days post-transportation. The ve rtical line separates baseline koi from transported koi.

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93 Figure 4-5. Koi behavior scores ( N = 25 koi observed for each metomidate treatment per observation time) from time 0 to 12 hours a nd 7 days post-transportation represented as a percentage of occurrences transported at various concentrations of metomidate. Categorical value scale; 1 = Very bad, >50% fish abnormal, 2 = Bad, >25% of fish abnormal, 3 = Average, 25% fish abnormal, and 4 = Excellent, 100% of fish normal. The vertical line separates baseline koi from transported koi.

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94 Figure 4-6. Koi behavior as a percentage of occurrence of sellable versus unsel lable tanks of fish ( N = 25 koi observed for each treatment per observation time) from time 0 to 12 hours and 7 days post-transportation. The ve rtical line separates baseline koi from transported koi.

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95 Figure 4-7. Koi activity scores ( N = 25 koi observed for each metomidate treatment per observation time) from time 0 to 12 hours a nd 7 days post-transportation represented as a percentage of occurrence. Categoric al value scale; 1 =Not active, 2 = Less active, 3 = Active, 4 = More active, and 5 = Very active/Hy peractive. Letters denote significantly different groupings ( P < 0.05). The vertical line separates baseline koi from transported koi.

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96 CHAPTER 5 THREESPOT GOURAMI PILOT STUDIES Objectives A series of pilot studies was conducted to determine 1) the metom idate concentration required to induce sedati on in threespot gourami Trichogaster trichopterus 2) if threespot gourami would survive 48 hour metomidate exposure and 3) the effects of metomidate on blood glucose levels. Threespot gourami may drown if the anesthetic drug induces a stage of anesthesia that prevents the fish from acce ssing the surface of the water for respiration. Therefore, it is important to determine the me tomidate concentration range that would induce sedation without interfering with the ability of the fi sh to maintain normoxia. Gourami Metomidate Range Finding Pilot Study Gouram i were subjected to simulated tran sportation and metomidate exposure. The gourami were observed for anesthetic plane that was induced after 24 hou rs in treatment and again at 48 hours in treatment. The gourami were then returned to their original tank on the recirculating tank system and observed for recovery. Methods Experimental Design Gouram i in a range of 50 mm TL ( N = 60) were selected for size and donated from a fish farm in Florida. Two gourami were placed into each tank and held for two weeks prior to experimentation. The gourami were fed Nutraf in Max Complete Flake Food for tropical fish (Hagen, Montreal, Canada) to satiation once daily. The recirculating tank system consisted of 30, 75.7-L tanks; a sump, a bubble-washed bead filter, an ultraviolet light sterilizer, and a fluidized bed filter that contained sand media. Each tank contained 37.8 L filtered well water, ha d an air stone, and drained to a common sump.

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97 Polystyrene partitions were placed between the ta nks to reduce the potential effect of visual stimuli from individuals in adjo ining tanks. The tanks were covered with a lid made of plastic mesh and PVC tubing to prevent the gourami from jumping out of the tanks. The gourami were held on a 12 h light: 12 h dark photoperiod. Dissolved oxygen (DO), pH, and temperature were analyzed directly from each tank weekly with an LDO digital meter (HQ20, H ach Company, Loveland, Colorado). Immediately afterwards, a water sample was collected and total ammonia (NH3 + NH4 +), nitrite (NO2 -), alkalinity, hardness, carbon dioxide (CO2), and salinity were analyzed within one hour of collection using a Hach Freshwater Fish Farmers Kit (FF-1A, Hach Company, Loveland, Colorado) and a salinity refractometer (A quatic Eco-Systems, Inc., Apopka, Florida). Water quality in the recirculating tank system was maintained at 0.3.4 mg/L total ammonia; unionized ammonia of 0.02.03 mg/L; 0.03 mg/L nitrite; pH of 8.1.2; temperature of 22.8.8C; alkalinity of 222 mg/L; hardness of 188 mg/L; carbon dioxide of 10 mg/L; and dissolved oxygen of 8.0.1 mg/L. Gourami were exposed to 0.25, 0.4, 0.6, 0.8, or 1.0 mg/L metomidate. There were two gourami per bag and one replicate for each treatme nt. A metomidate stock solution of 1 mg metomidate per 1 mL distilled water was used to dose 3.78 L shipping water in square-bottom, quarter-size, plastic shipping bags (191 mm x 165 mm x 500 mm) with one of the five metomidate concentrations. Each bag with sh ipping water and metomidate was then mixed for 30 seconds. Simulated Transportation The gourami were placed into square-bottom quarter-size, plastic shipping bags (191 mm x 165 mm x 500 mm) with 1.0 L aerated meto midate dosed well water with 4 g/L salt (NaCl) (Morton, Chicago, Illinois). The bags were then filled with oxygen gas, closed with a

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98 rubber band, and placed into a polystyrene, square, single shipping box (432 mm x 432 mm) with a lid. The two boxes were stacked side-by-side on a wood pallet. An air compressor (Sears Craftsman hp, 22.7 L, Hoffman Estates, Illinois) was also placed on the pallet to simulate movement of shipping (Figure 41). Every 16 minutes the air co mpressor shook the pallet for 30 seconds for the duration of the experiment. In addition, an air pump (Supreme Dynamaster air pump, Monroeville, Pennsylvania) was placed on the top of each box to simulate the movement of transportation; the pumps ran fo r the duration of the experiment. Observations Gouram i were observed for stage of anesthes ia in each bag at 24 hours and again at 48 hours of simulated tran sportation. Following 48 hours in si mulated transpor tation, the gourami were placed back into their original tank on the recirculati ng tank system and observed for recovery of normal behavior. Results Gouram i exposed to metomidate concentratio ns of 0.25 and 0.4 mg/L were maintained in light sedation for the duration of time in simula ted transport (Table 5-1). Both gourami sedated with 0.25 mg/L metomidate and one gourami sedate d with 0.4 mg/L metomi date recovered from the effects of sedation within one hour (Table 5-1). One gourami sedated with 0.4 mg/L metomidate had difficulty surfacing to respire and was placed in a net at the surface of the water for 96 hours before full recovery (Table 5-1). This action was taken to determine the length of time required for the fish to recover, if at all. Gourami that were in metomidate concentrations greater than 0.4 mg/L also had difficulty surfacing to respire and were plac ed into nets at the surface of the water to observe the time required for normal behavior to return, if at all. At a metomidate concentration of 0.6 mg/L the gouram i were in light narcosis, and at 0.8 mg/L

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99 metomidate the gourami were in deep narcosis after 24 and 48 hour meto midate exposure. Of two gourami exposed to 1.0 mg/L metomidate, one died during the first 24 hours of metomidate exposure in simulated transport and the other was observed to be in d eep narcosis. This fish died 30 minutes after return to the original tank Discussion Metom idate induced sedation in threespot gourami within the recommended dosage range of 0.25.0 mg/L. Threespot gourami exposed to metomidate concentrations of 0.1, 0.2, 0.3, and 0.4 mg/L were maintained in a sedative stage of anesthesia for the duration of the 48 hour pilot study. Gourami exposed to metomidate concentrations higher than 0.4 mg/L entered light narcosis, and exposure to 1.0 mg/L metomidate resulted in mortality. Therefore, metomidate concentrations of 0.1, 0.2, 0.3, and 0.4 mg/L were used for the threespot gourami experiment. Holding fishes in a net at th e surface of the water is a prac tice frequently used by retail fish stores for fishes that are perceived to be under duress upon arrival. The fishes are so held until normal behavior is observed. However, th e use of this technique in this experiment affected the evaluation of potential additional deleterious effects, including mortality, and affected recommendations for specific concentrati ons of metomidate that were utilized with threespot gourami. Ross and Ross (1999) state that obligate air br eathing fishes are a challenge to sedate because they must respire atmospheric oxygen. In this study, metomidate was effectively absorbed across the gills and th reespot gourami were not observed to be difficult to sedate. Gourami Blood Glucose Pilot Study The objective of this study wa s to de termine the effect of metomidate sedation on blood glucose levels in threespot gourami exposed to simulated transportation for 48 hours.

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100 Methods Experimental Design The gourami were the sam e fish as previ ously described on page 96, Experimental Design. There were 7 days between the previous experiment and this experiment. Gourami were maintained in the same recirculating tank system previously described and water quality was analyzed as before (pag es 96 Experimental Design). Water quality in the recirculating tank system was maintained at 0.3.4 mg/L total ammonia; unionized ammonia of 0.03.04 mg/L; 0.03 mg/L nitrite; pH of 8.1.2; temperature of 22.8.8C; alkalinity of 222 mg/L; hardne ss of 188 mg/L; carbon dioxide of 5 mg/L; and dissolved oxygen of 7.5.2 mg/L. The gourami were exposed to simulated transpor tation in metomidate concentrations of 0, 0.1, 0.2, 0.3, and 0.4 mg/L. There were two gourami per bag and three replicates for each treatment. A metomidate stock solution (page 97 Experimental Design) was used to dose a total of 5 L shipping water in a square-bottom, half -size, plastic shipping bag (406 mm x 203 mm x 558 mm) to the appropriate metomidate concen tration. Each bag of shipping water and metomidate was then mixed for 30 seconds. These bags were then used to fill individual shipping bags. Another group of gourami that were not transported or ex posed to metomidate represented a baseline group. Simulated Transportation The gourami were placed into a square-bottom quarter-size, plasti c shipping bag (191 mm x 165 mm x 500 mm) with 1.0 L aer ated metomidate dosed shippi ng water with 4 g/L salt. The bags were then filled with oxygen gas, cl osed with a rubber band, and placed into a polystyrene, square, single shipping box (432 mm x 432 mm) with a lid. A six minute time

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101 stagger in placing gourami into bags between each tank allowed for blood sampling to be completed. The five boxes were stacked four boxes side -by-side on a wood pallet; the fifth box was centered on top of the other four boxes. An air compressor (Sears Craftsman hp, 22.7 L, Hoffman Estates, Illinois) was also placed on the pallet to simulate the movement of transportation (Figure 4-1). Th e air compressor shook the pallet for 30 seconds every 16 minutes for the duration of the experiment. In addi tion, two air pumps (Supreme Dynamaster air pump, Monroeville, Pennsylvania) were placed on the top box to simulate the movement of transportation, and they both ran the duration of the experiment, 48 hours. Blood Collection and Blood Chemistry The gourami were placed into dorsal recum bency and 0.3 mL of blood was collected from the cardiac or duct of Cuvier site using a 25-G needle fitted on a 1-mL syringe containing about 0.06 mL sodium heparin (Baxter Healthcare Corporation, Deerfield, Illinois). After blood collection, the gourami were euthanized in a 1000 mg/L MS-222 bath buffered with 2000 mg/L sodium bicarbonate (NaHCO3). To standardize the sampling protocol, gourami were sampled within 4 minutes from when a net first entered the tank or bag. Blood glucose levels were immediately analyzed using a hand-held Ascensia ContourTM glucose meter (Bayer Healthcare, Morrist own, New Jersey). The meter required 0.6 L of whole blood, 15 seconds for analysis, and ha d a detectable range of 10-600 mg/dL. If 0.3 mL total volume of blood and heparin was collected before the 4-minute time limit expired, the sample was immediately submitted for blood gluc ose analysis. If 0.1.3 mL total volume was collected within 4 minutes, blood glucose was analy zed from the collection attempt. However, if less than 0.1 mL blood was collected or no bloo d was collected before the 4 minutes expired,

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102 sampling ceased and the gourami was euthanized. The blood glucose levels were then corrected for heparin dilution. Blood glucose data were anal yzed using one-way ANOVA at = 0.5 followed by Tukeys HSD comparison of means in JMP 5.1 Statistical Discovery Software (SAS Institute, Cary, North Carolina). For blood gl ucose levels that were below the minimum level of detection for the meter (i.e., <10 mg/dL), a value of 5 mg/dL was arbitrarily assigned in order to determine the significance of blood glucose levels among the various concentra tions of metomidate. Results Gouram i exposed to 0.2 mg/L metomidate ha d significantly higher bl ood glucose levels (49.5 mg/dL) compared to baseli ne gourami (16.3 mg/dL) (ANOVA: F = 3.38; df = 5, 29; P = 0.02) (Figure 5-1). Blood glucose levels of gourami transpor ted with 0, 0.1, 0.3, and 0.4 mg/L metomidate (25.0.7 mg/dL) were not signifi cantly different from baseline gouramis. However, blood glucose levels from all gourami subjected to simulated transportation were not significantly different from each other (25.0.4 mg/dL). Discussion The gourami exposed to 0.2 m g/L metomidate had a physiological response to stress which suggests that this concentrat ion of metomidate is not effec tive at inhibiting an increase in blood glucose levels. However, the blood glucose levels of all gouramis exposed to simulated transport were not different; further testing is needed to clarify the metomidate concentration required to reduce an increase in gourami blood glucose levels. The lack of appreciable difference in blood glucose levels between ba seline gourami and gourami exposed to 0 mg/L metomidate may have been due to stress from th e activity in the laborator y. However, there are no baseline blood glucose data on threespot gourami availa ble for data comparison.

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103 Table 5-1. Gourami range fi nding pilot study observations. a Metomidate (mg/L) Time Observations 0.25 24 Hours in treatment Both fish light sedation 0.40 24 Hours in treatment Both fish light sedation 0.60 24 Hours in treatment One fish light sedation, one fish light narcosis 0.80 24 Hours in treatment Bo th fish deep narcosis 1.00 24 Hours in treatment One fish deep narcosis, other fish dead 0.25 48 Hours in treatment Both light sedation 0.40 48 Hours in treatment Both light sedation 0.60 48 Hours in treatment One fish light sedation, one light narcosis 0.80 48 Hours in treatment Bo th fish deep narcosis 1.00 48 Hours in treatment One fish dead, other fish deep narcosis 0.25 1 Hour in recovery Both fish recovered 0.40 1 Hour in recovery One fish re covered, other fish light sedation 0.60 1 Hour in recovery Both fish light sedation 0.80 1 Hour in recovery Both fish light sedation 0.40 2 Hours in recovery One fish recovered, other fish light sedation 0.60 2 Hours in recovery Both fish light sedation 0.80 2 Hours in recovery Both fish light sedation 0.40 3 Hours in recovery One fish recovered, other fish light sedation 0.60 3 Hours in recovery One fish recovered, one fish light sedation 0.80 3 Hours in recovery Both fish light sedation 0.40 4 Hours in recovery One fish recovered, one fish light sedation 0.60 4 Hours in recovery One fish recovered, one fish light sedation 0.80 4 Hours in recovery Both light sedation 0.40 24 Hours in recovery One fish re covered, other fish light sedation 0.60 24 Hours in recovery Both fish recovered 0.80 24 Hours in recovery Both fish recovered 0.40 27.5 Hours in recovery One fish re covered, other fish light sedation 0.40 31 Hours in recovery One fish re covered, other fish light sedation 0.40 48 Hours in recovery One fish re covered, other fish light sedation 0.40 72 Hours in recovery One fish re covered, other fish light sedation 0.40 96 Hours in recovery Both fish recovered a The gourami were exposed to concen trations of 0.25, 0.40, 0.60, 0.80, and 1.0 mg/L metomidate and subjected to 48 hours simulated transportation. Gourami were observed for stage of anesthesia at 24 and 48 hours metomida te exposure and for behavior during recovery.

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104 Figure 5-1. Gourami blood glucose levels after 48 hours metomidate exposure. Letters denote significantly different groupings ( P < 0.05) with standard error bars. The vertical line separates baseline gourami from gourami exposed to simulated transportation.

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105 CHAPTER 6 THREESPOT GOURAMI EXPERIMENT Introduction Aquacultured ornam ental fishes from Florid a contribute to the st ates economy. In 2005, the agricultural census conducted by the Unite d States Department of Agriculture National Agricultural Statistics Service (USDA NASS 2006) reported an a quaculture farm-gate value in Florida of $75 million, of which, $32.4 million was generated by Floridas tropical fishes sales. Approximately 1 million gourami Trichogaster sp. are reportedly sold from one large Florida wholesale distributor of ornament al fishes annually with a farm gate value of $0.25.30 per fish (wholesaler, persona l communication). Preservation of the condition of fishes (i.e., quality) post-transporta tion is essential to profitability. Transportation and handling are stre ssful to fishes and may affect marketability (i.e., appearance, behavior, and activity level) and profitability through the effects of trapping, netting, and crowding fishes prior t o, during, and after transportation. Stress is defined as the physiological respons e to sustain homeostasis in response to a stimulus (Barton 1997). An acute stressor, such as transportation of fishes, initiates the production of glucocorticoid hor mones that leads to an increase in blood glucose levels (Mommsen et al. 1999, Al-Kindi et al. 2000). Therefore, blood gl ucose elevation is considered an indicator of the physiologica l stress response (Mazeaud et al. 1977) At this time there are no studies of the effect of transportation on blood glucose levels in threespot gourami Trichogaster trichopterus However, several studies of food fishes have demonstrated an increase in blood glucose levels in response to acute stress in Chinook salmon Oncorhynchus tshawytscha (Sharpe 1998) and coho salmon Oncorhynchus kisutch (Wedemeyer 1972), to net confinement in rainbow trout Oncorhynchus mykiss (Woodward and Strange 1987), to lowered water volume in

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106 the striped bass Morone saxatilis (Davis and Griffin 2004), and to transportation in red drum Sciaenops ocellatus (Robertson et al. 1988, 1987) and spotted sorubim Pseudoplatystoma corruscans (Fagundes et al. 2008). Physical trauma and advers e physiological response to transportation stress may be reduced with an anesthetic. An anesthetic affects the entire body in a dose-dependent or exposure-related continuum of responses through reversible depression of the central nervous system (Summerfelt and Smith 1990, Steffey 1995). A commonly used anesthetic scheme is Stoskopfs (1995) four stages of anesthesia that includes sedation, narcos is, light to surgical anesthesia, and medullary collapse. Sedation is the first stage of anesthesia and is the most desirable level for fish transportation. Under sedation fishes may have varied responses to stimuli, and motion and respiratory rate are re duced; however, equilibrium may remain normal. Fishes in the suborder Anabantoidei have a labyri nth organ that functions similar to a terrestrial lung; this enables the fishes to respire ai r above the water surface (Stoskopf 1993a, Graham 1997, Barton 2007). The anabantoid threespot gourami is an obligat e air-breather, and these fish may suffocate without access to the water surface (Von Ramshorst 1981, Cole et al. 1999, Moyle and Cech 2000). Therefore, threespot gourami shoul d be transported no deep er than a sedative stage of anesthesia. Any anesthetic agent that induces narcosis or su rgical anesthesia may interfere with osmoregulation (Wedemeyer 1996a, Ross and Ross 1999), and may result in fishes that sink to the bottom of the shipping cont ainer and may become hypoxic due to impaired ability to respire at the surface of the water. In addition, loca lized areas of low dissolved oxygen (DO) may also occur (Ross and Ross 1999). Metomidate hydrochloride (hereafter referred to as metomidate) is an imidazole-based, hypnotic drug that is effective for anesthesia but not analgesia (Branson and Booth 1995, Treves-

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107 Brown 2000). Metomidate, trade name AquacalmTM (Syndel Laboratories, Ltd., Qualicum Beach, British Columbia, Canada), is currently ap proved for use in Canada as an anesthetic in aquarium and non-food fishes in the order Silu riformes (i.e., catfish) and in the families Cyprinidae (e.g., carp, goldfish), Poeciliidae (e.g. live-bearers), Centra rchidae (e.g., sunfish, bass), Cichlidae (i.e., cichlids), and Pom acentridae (i.e., saltwater damselfish). Metomidate is thought to suppr ess the 11hydroxylat ion of cholesterol that is required for production of plasma cortisol in the interrenal tissue (Wada et al. 1988, Olsen et al. 1995). Indeed, in a study with threespot gouramis, Crosby et al. (2006c ) reported significantly lower plasma cortisol levels in gourami exposed to 0.8 mg/L metomidate afte r acute handling stress compared to untreated control i ndividuals. Increased cortisol production leads to an increase in blood glucose levels through glycogenesis a nd gluconeogenesis (Mommsen et al. 1999); therefore blood glucose elevation should be reduced as well. Additionally, metomidate does not accumulate with long exposure time, and has little effect on water chemistry (R. Bradshaw, Syndel Laboratories Ltd., persona l communication). For example, exposing southern platyfish Xiphophorus maculatus to 1.0 mg/L metomidate had no effect on water pH, total ammonia, or carbon dioxide during 48 hours simulated transp ortation (Guo et al. 1995a). Additionally, oxygen consumption was decreased in southern pl atyfish exposed to 1.0 mg/L metomidate (Guo et al. 1995b). Despite the reported benefits, metomidate may have deleterious effects, including mortality. Southern platyfish exposed to 1.0 mg/L metomidate for 48 hours had 11% mortality compared to 6% in untreated co ntrol individuals (Guo et al. 1995a ). Also, 5 days after exposure metomidate treated southern plat yfish had 42% mortality compared to 12% in untreated control individuals (Guo et al. 1995a). In addition, sout hern platyfish exposed to metomidate had an

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108 increase in oxygen consumption as temperature was increased from 20C to 30C compared to untreated control individuals (Guo et al. 1995b). Hill et al. (2005) also noted that some rainbow sharks Epalzeorhynchos frenatum exposed to metomidate prior to topical gill application of a spawning hormone would intermittently react strongly to external s timuli (J. E. Hill, University of Florida, personal communication). Additiona lly, Massee et al. (1995) reported long induction and recovery times in larval goldfish Carassius auratus under metomidate anesthesia, and concluded that metomidate at 6 mg/L was not an effective anesthetic for these fish. This study investigated metomidate sedation in threespot gourami transported for approximately 24 hours. Threespot gourami was chosen for this study because of its economic value and because obligate air-breathing fishes ar e reportedly difficult to sedate (Ross and Ross 1999). The need to respire atmospheric oxygen (G raham 1997) makes the determination of the optimal metomidate concentration critical for threespot gourami. The specific objectives were 1) to measure blood glucose levels post-trans portation and 2) to determine the effect of metomidate sedation on marketability as determined through vi sual assessment of ap pearance, behavior, and activity levels post-trans portation. The objectives were tested through two separate experimental components: blood glucose and marketability. Methods Experimental Design Threespot g ourami in a size ra nge from 50 mm to 76 mm TL ( N = 330) were donated from a fish farm in Florida and were placed in a recirculating tank system that consisted of 30, 75.7-L tanks, a sump, a bubble-washed bead filter, an ultraviolet light ster ilizer, and a fluidized bed filter that contained sand media. A polystyre ne partition was placed between tanks, and each tank was filled with recirculated filtered water, aerated with an air stone, and drained into a common sump. The tanks were covered with a lid made of plastic mesh and PVC tubing to

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109 prevent the gourami from jumping out of the ta nks. The photoperiod was 12 h light: 12 h dark except for the day of the experiment in which th e lights were on during data collection for a total of 36 hours. Salt (NaCl) (Morton, Chicago, Il linois) was maintained in the system at 3.0.5 g/L All gourami were fed to satiation once da ily with Nutrafin Max Complete Flake Food for tropical fishes (Hagen, Montreal, Canada). For the blood glucose component of the experiment, gourami were placed into each of th e 30 tanks containing 37.8 L well water. In the marketability component of the experiment, gourami were placed into each of 30 tanks containing 18.9 L of well water. Dissolved oxygen, pH, and temperature were measured with an LDO digital meter (HQ20, Hach Company, Loveland, Colorado) week ly from each tank. Immediately after the DO, pH, and temperature were recorded a water sample was collected from each tank and analyzed within an hour of co llection for total ammonia (NH3 + NH4 +), nitrite (NO2 -), alkalinity, hardness, and carbon dioxide (CO2) with a Hach Freshwater Fi sh Farmers Kit (FF-1A, Hach Company, Loveland, Colorado). Salinity was measur ed with a salinity refractometer (Aquatic Eco-Systems, Inc., Apopka, Florida). Recirculating tank system water quality was maintained at 0.5 mg/L total ammonia; uni onized ammonia of 0.09 mg/L; 0.7 mg/L nitrite; pH of 7.5 8.5; temperature of 22.9.7C; alkalinity of 188 mg/L; hardness of 188 mg/L; carbon dioxide of 5 mg/L; and di ssolved oxygen of 7.6.9 mg/L. The gourami were transported at five con centrations of metomidate: 0, 0.1, 0.2, 0.3, and 0.4 mg/L. There were five replicates for each treatment. In addition to the metomidate treatments, there was a baseline group of threes pot gourami that were not transported or exposed to metomidate. Baseline and experimental groups were randomly assigned to each tank. For the blood glucose component of the experiment there were six gourami per tank. In the

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110 marketability component of the experiment ther e were five gouramis per tank Treatment concentrations were based on p ilot studies (Chapter 5) and we re within the manufacturers recommended dose for sedation of 0.1.0 mg/L Shipping water was dosed with a metomidate stock solution of 1 mg metomidate per 1 mL distilled water. Each square-bottom, qua rter-size, plastic shipping bag (191 mm x 165 mm x 500 mm) was filled with 1 L water, metomidate solution, 4 g/L salt, and then mixed for 30 seconds. In Florida, medium-size gour ami (i.e., ranging from 44.5.8 mm TL) are transported with 125 fish per full-size, squa re-bottom, plastic shipping bag (394 mm x 368 mm x 572 mm) with 7.57 L of shipping water, a nd large-size gourami (i.e., ranging from 50.8 63.5 mm TL) are shipped 75 fish per full-size, square-bottom, plastic shipping bag with 7.57 L of water (Crosby et al. 2006b). The gourami were transported at half the typical shipping density based on water volume. Transportation In the blood glucose com ponent of the experiment there was a time stagger of 6 minutes between each tank to ensure completion of blood co llection. For the market ability component of the experiment, the bags of gourami were set in to boxes with no delay in bagging time. The bags were filled with oxygen gas, closed with a rubber band, placed into a polystyrene, square, single shipping box (432 mm x 432 mm) with a lid, and each box was placed inside an outer cardboard box for labeling. Bags were randomly assigned to a box; there were four bags per box, and empty air filled bags were used as place holders. Subsequently, boxes were transported via truck for approximately 2.5 hours from Gainesville, Florida, to a distribut ion facility of ornamental fish es in Gibsonton, Florida. The boxes of gourami were transported the followi ng morning about 12 hour s later by truck to Tampa International Airport in Tampa, Florida, a nd flown via domestic airline to North Carolina.

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111 The boxes were then returned vi a domestic airline to the Ga inesville Regional Airport in Gainesville, Florida, that same af ternoon and driven via truck back to the research laboratory at the University of Florida, Program in Fisheries and Aquatic Sciences. Total transportation time was about 24 hours. Blood Collection and Blood Glucose One gourami per tank was sam pled at each sample time. Blood was collected from the cardiac or duct of Cuvier site at four sampling times post-tr ansport: 0, 2, 6, and 12 hours. The cardiac and duct of Cuvier sites are both cons idered a cardiac blood co llection site (Houston 1990). To obtain blood, gourami were placed into dorsal recumbency, and blood samples of 0.3 mL were collected with a 25-G needle fitte d on a 1-mL syringe c ontaining about 0.06 mL sodium heparin (Baxter Healthcare Corporation, Deerfield, Illinois). At time 0 hour post-transportation, a bag was opened, and a gourami was removed and immediately sampled. The remaining gourami in the bag were placed into the original tank on the recirculating tank system for subsequent sampling. The gourami were sampled within 4 minutes from when a net first entered the bag or tank to standardize the sampling time. If 0.3 mL total volume blood and heparin was collected before the 4 minutes expired blood glucose was immediately analyzed. If 0.1.3 mL total volume was collected within 4 minutes, the blood glucose was analyzed from the collection a ttempt. However, if less than 0.1 mL total volume was collected when the 4 minutes expired, sampling ceased. After sampling, the gourami were euthanized in a solution containing 1000 mg/L MS-222 (Western Chemical, Ferndale, Washington) buffered with 2000 mg/L sodium bicarbonate (NaHCO3) (Arm & Hammer, Princeton, New Jersey). Immediately after blood was collected, blood glucose was analyzed with a hand-held Ascensia ContourTM glucose meter (Bayer Healthcare, Morristown, New Jersey). The meter

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112 required 0.6 L whole blood, analyzed a sample in 15 seconds, and had a detectable range of 10600 mg/dL. The blood glucose levels were then corrected for heparin dilution. The differences in mean blood glucose levels were analyzed us ing one-way analysis of variance (ANOVA) with a Type I error rate ( ) of 0.5 followed by Tukeys HSD comparison of means in JMP 5.1 Statistical Discovery Software (SAS Institute, Cary, North Carolina). For blood glucose levels that were below 10 mg/dL, the minimum level of detection for the meter, a value of 5 mg/dL was arbitrarily assigned to determine the significance among bloo d glucose levels. Appearance, Behavior, and Ac tivity Level Observations The appearance, behavior, and activity leve l of gouram i in each tank were assessed by four judges using a ranked, categorical valu e system (see Table 6-1). The gourami were observed at six times post-transporta tion: 0, 2, 4, 6, and 12 hours and 7 days. Appearance was judged using visual charac teristics including coloration, scale loss, ragged fins, hemorrhaging, and exophthalmia. Scor es of 1 to 2 denoted unsellable fish and scores of 3 to5 were sellable fish. Behavior was observed for characteristics such as hiding, isolation from tank mates, hanging in the wate r column, piping, gaspi ng, spinning, flashing, and loss of buoyancy. Scores of 1 to 2 were unsellable and 3 to 4 were sellable fish. Activity level was observed for whether gourami were not active, normally active, or very active. Activity level scores ranged from 1 that designated not active to a score of 5 that denoted very active. Appearance, behavior, and activity data were organized by rank sum. A Kruskal-Wallis (K-W) was performed at = 0.05 using JMP 5.1 Statistical Disc overy Software (SAS Institute, Cary, North Carolina) to determine differences in the mean rank of scores. If significant differences were observed, the data were then analyzed by Dunns comparison of means to determine differences among treatments (Hollander and Wolfe 1973). Due to the conservative nature of the Dunns comparison of means test, = 0.15 was used (Hollander and Wolfe 1973).

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113 In addition, the significance between proportions of tanks that had sellable gourami to tanks that had unsellable gourami was evaluated using one-w ay analysis of variance (ANOVA) with a Type I error rate ( ) of 0.5 followed by Tukeys HSD comparison of means in JMP 5.1 Statistical Discovery Software (SAS Institute, Ca ry, North Carolina). Data were arcsine squareroot transformed in order to meet normality assumptions of the ANOVA (Microsoft Office Excel 2003). Shipping Water Physicochemistry After arrival of transported gouram i to the laboratory, dissolved oxygen, pH, and temperature were measured with an LDO digita l meter and a water sample was collected from each shipping bag as it was opened. The water samp les were stored in a re frigerator at 4C for approximately 20 hours and then total ammonia and carbon dioxide were tested with a Hach Freshwater Fish Farmers Kit. The shipping water physicochemistry data were analyzed using one-way analysis of variance (ANOVA) with a Type I error rate ( ) of 0.5 followed by Tukeys HSD comparison of means in JMP 5.1 Statistical Discovery Software (SAS Institute, Cary, North Carolina). For dissolved oxygen values that were above the ma ximum level of detection for the meter (i.e., greater than 20 mg/L), a value of 21 mg/L was ar bitrarily assigned to determine the significance among metomidate concentrations. Results Blood Glucose At tim e 0 hour post-transportation gourami transported with 0.2 mg/L metomidate had significantly lower blood glucose levels (94.7 mg/dL) compared to those transported with 0.4 mg/L metomidate (225.1 mg/dL) (ANOVA: F : = 2.97; df = 5, 25; P = 0.03) (Figure 6-1). Also, blood glucose levels of gourami transported with 0, 0.1, 0.2, and 0.3 mg/L metomidate (94.7

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114 150.3 mg/dL) were not significantly different from each other (Figure 6-1). Blood glucose levels of gourami transported with 0.1 mg/L metomidate (124.6), 0.2 mg/L meto midate (94.7 mg/dL), and 0.3 mg/L metomidate (121.6 mg/dL) were not si gnificantly different from levels of baseline gourami (18.2 mg/dL) (Figure 6-1). At two hours post-transportation, gourami transported at 0, 0.1, 0.2, and 0.3 mg/L metomidate had significantly lower blood glucose levels (98.1.4 mg/dL) than those transported with 0.4 mg/L metomidate (217.9 mg/dL) (ANOVA: F = 7.33; df = 5, 30; P = 0.0001) (Figure 6-1). At time 12 hours post-transportation g ourami transported with 0.2 mg/L metomidate (29.2 mg/dL) had signif icantly lower blood glucose levels than those transported with 0.4 mg/L metomidate (66.3 mg/dL) (ANOVA: F = 3.21; df = 5, 28; P = 0.02). Appearance, Behavior, and Ac tivity Level Observations There were no significant diffe rences in overall appearance of goura mi (Figure 6-2). At time 6 hours post-transportation gourami transporte d with 0 and 0.4 mg/L metomidate had higher percentages of sellable fish based on appearance (ANOVA: F = 4.39; df = 5, 24; P = 0.006) (Figure 6-3). Also, at time 12 hours post-tran sportation gourami tran sported with 0 mg/L metomidate had a lower percentage of sellabl e fish compared to baseline gourami based on appearance (ANOVA: F = 4.93; df = 5, 24; P = 0.003) (Figure 6-3). At time 0 hour posttransportation gourami transported with 0.3 mg/L metomidate had significantly lower behavior scores than baseline gourami; however, beha vior of all transported gourami were not significantly different (KW: H = 3.23; df = 5, 24; P = 0.02) (Figure 6-4). Also at time 0 hour post-transportation, the percentage of tanks of sellable gourami transported with 0.3 mg/L metomidate was significantly lower than baseli ne tanks of gourami based on behavior (Figure 65). There were no significant differences in activity level of gourami (Figure 6-6).

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115 Shipping Water Physicochemistry In the blood glucose com ponent of the experiment, there were no significant differences in dissolved oxygen, total ammonia, carbon dioxide, or pH (Table 6-2). The temperature of gourami transported with 4.0 mg/L metomidate (22.1 C) was significantly higher than that of all other transported gouram i (21.7.8C) (ANOVA: F = 6.36; df = 4, 20; P = 0.0018) (Table 62). For the marketability component of the expe riment, there were no significant differences in shipping water physicochemistry parameters te sted (i.e., dissolved oxygen, total ammonia, carbon dioxide, pH, and temperature) (Table 6-2). Discussion Gouram i are economically important to the Fl orida aquaculture indus try. Transportation of these fishes is stressful and may have a dverse effects on their ove rall marketability posttransport. The stress of transportation increase s the production of cortis ol and glucose, both of which are considered indicators of stress (Ma zeaud et al. 1977, Wedemeyer 1996a). In addition, there are several other biological indicators of stress such as blood hematocrit, blood osmolality, blood leucocrit, and interrenal hypertrophy (Wedemey er et al. 1990). For these reasons, it is important to study transportation methodology that will improve th is component of ornamental fish aquaculture. Sedatives may be used to reduce the deleterious effects associated with handling and transportation. However, there is little information about transporting threespot gouramis with metomidate. Because of this lack of information and the fact that the threespot gourami is an obligate air-breathing fish, it is e ssential to determine the correct metomidate concentration that will not impair the ability of th e fish to respire at the surface of the shipping water. In this study at time 0 ho ur post-transportati on, gourami transported with 0.1, 0.2, and 0.3 mg/L metomidate had blood glucose levels that were not different from baseline fish. In

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116 addition, at these metomidate c oncentrations, the results appear ed to exhibit a non-significant, non-linear trend of lowered blood gl ucose levels compared to othe r metomidate concentrations tested. Based on this trend, the use of 0.1, 0.2, and 0.3 mg/L metomidate during transportation may be good concentrations for inhibiting a ri se in threespot gourami blood glucose levels. Similar to the results of the present study, Davi s and Griffin (2004) repor ted that hybrid striped bass ( M. saxatilis x M. chrysops ) sedated with 1.5 mg/L metomi date had blood glucose levels comparable to un-sedated, cont rol individuals. Blood glucose levels in low-water stressed channel catfish Ictalurus punctatus exposed to 8 mg/L metomidate we re lower than the levels of stressed, control fish as well (B osworth et al. 2006). In anot her study with threespot gourami, Crosby et al. (2006c) concluded that exposure to 0.8 mg/L metomidate af ter acute handling stress lowered the gouramis response to the stressor as i ndicated by an inhibition of increased plasma cortisol levels, a precursor that stimulates glucose production. An important consideration is that a higher metomidate concentrati on of 0.4 mg/L may be contraindicated for transportation of threespot gouramis. At times 0 and 2 hours posttransportation, gourami transporte d with 0.4 mg/L had the highest mean blood glucose levels compared to other transported individuals, including the control. These results suggest that the gourami treated with 0.4 mg/L metomidate were more stressed as compared to the gourami at the other metomidate treatments. Transportation of threespot gourami was stre ssful as indicated by higher blood glucose levels at time 0 hour post-transportation in cont rol gourami compared to baseline gourami that were not transported. These results are consiste nt with reported increa sed blood glucose levels following handling and transportation in the blackeye thicklip wrasse Hemigymnus melapterus (Grutter and Pankhurst 2000). Fagundes et al. (2 008) also concluded that spotted sorubim

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117 Pseudoplatystoma corruscans had increased blood gl ucose levels compared to control fish in response to transportation stress. In this study, the use of metomidate during transportation had no apparent beneficial or detrimental effects on gourami marketability. However, at time 0 hour post-transportation, gourami transported with 0.3 mg/L metomidate were less marketable than baseline fish based on behavioral observations. Thus, although there we re no statistically sign ificant differences in these observations among transpor ted fish, the use of 0.3 mg/L ma y have a detrimental impact on behavior. While some researchers have stated that th e use of a sedative in shipping water will reduce oxygen consumption and total ammonia and carbon dioxide production (Summerfelt and Smith 1990, Wedemeyer 1996a), the dissolved ox ygen, total ammonia, carbon dioxide, and pH from shipping water in this study were not diffe rent among the metomidate treatments. The temperature differences that were less than 0.5C in the blood chemistry component of the experiment likely had no effect on the physiologi cal response to transportation. Also, there was no difference in the water physicochemistry parame ter most affected by temperature, dissolved oxygen. In the blood glucose component of the experiment, two mortalities were observed in gourami transported with 0.4 mg/L metomidate. Ho wever, in the marketability component of the experiment, there were no mortalities noted in gourami transported at the same metomidate concentration. A possible explan ation for this disparity is th e difference in the number of gourami (5 vs. 6) transported in each bag which may have affected the uptake of metomidate. In conclusion, a metomidate concentrati on of 0.1, 0.2, and 0.3 mg/L during transportation of threespot gouramis appeared to inhibit an increase in blood glucose levels in this study.

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118 However, the use of 0.3 mg/L metomidate may have detrimental effects on gourami behavior. Therefore, the best metomidate concentrations for glucose inhibition of transported threespot gourami in this study were 0.1 and 0.2 mg/L. Also, gourami transported with 0.4 mg/L metomidate had high blood glucose levels, and consequently 0.4 mg/L metomidate may have negative effects for transportation of this specie s. It is important to note the lowered blood glucose levels alone do not confirm that the gourami were not stressed; but rather their ability to respond to transport stress may have been re duced (Alistair Webb, Univ ersity of Florida, personal communication). Further metomidate transportation studies with additional biological indices of stress such as plasma cortisol, blood hematocrit, blood osmolality, and blood leucocrit, as well as transport densities typical of the commercial industry, may result in a different conclusion than presented here.

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119 Table 6-1. Evaluations of gouram i appearance, behavior, and activ ity level for observations of fish marketability post-transportation. Score Criteria Considerations Appearance 1 Very Bad-Unsellable More than 50% of gourami abnormal with minor to moderate damage of eyes/body/fins Coloration, hemorrhaging, scale-loss, deformities, ragged/torn fins, clamped fins, exophthalmia, cloudy eyes, lesions, parasites 2 Bad-Unsellable 25% of gourami abnormal with minor to moderate damage of eyes/body/fins 3 Average Less than or equal to 25% of gourami abnormal, eyes/body/fins damage minor 4 Good Gourami overall look normal, very little eyes/body/fins abnormalities 5 Excellent Gourami overall extremely healthy looking, vibrant color, eyes/body/fins superior Behavior 1 Very Bad-Unsellable More than 50% of gourami abnormal Piping, gasping, spinning, flashing, loss of buoyancy control, isolated/hiding, located in abnormal part of water column 2 Bad-Unsellable % of gourami abnormal 3 Average Less than or equal to 25% of gourami abnormal 4 Excellent All gourami behaving alert Activity Level 1 Not Active Overall activity 2 Less Active 3 Active 4 More Active 5 Very Active

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120Table 6-2. Mean (SD) gourami shipping wate r physicochemistry parameters. Different le tters indicate signif icant differences a cross rows ( P < 0.05). Metomidate (mg/L) Experiment Component Parameter a 0 0.1 0.2 0.3 0.4 F-ratio b Pr > F Blood Glucose DO 17.23.3 17.12.1 18.52.4 17.12.37 17.42.2 0.27 0.89 TA 18.60.5 19.64.6 20.63.1 19.82.6 17.26.4 0.55 0.70 CO2 80.06.7 78.04.0 58.03.5 67.00.2 97.04.6 1.76 0.18 pH 6.50.1 6.50.0 6.50.0 6.50.0 6.50.1 0.59 0.68 Temp C 21.8.1 z 21.8.2 z 21.7.1 z 21.8.1 z 22.1.2 y 6.36 0.0018 Marketability DO 21.00.0 20.02.2 21.00.0 21.00.0 21.00.0 1.00 0.43 TA 8.81.5 9.22.2 8.40.5 8.61.1 9.21.3 0.31 0.87 CO2 41.012.4 41.011.0 39.05.5 42.011.5 41.011.9 0.06 0.99 pH 6.50.1 6.50.0 6.50.0 6.50.0 6.50.1 0.59 0.68 Temp C 20.60.1 20.70.3 20.90.5 20.90.2 20.50.3 1.46 0.25 a DO, TA, and CO2 are in mg/L; temp C = temperature in degrees Celsius. b Numerator df = 4, denominator df = 20.

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121 Figure 6-1. Mean blood glucose levels of threespot gourami at times 0, 2, 6, and 12 hours posttransportation. Letters denote sign ificantly different groupings ( P < 0.05) with standard error bars. The vertical line separates base line gourami from transported gourami.

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122 Figure 6-2. Gourami appearance scores ( N = 25 gourami observed for each metomidate treatment per observation time) from time 0 to 12 hours and 7 days posttransportation represented as a percentage of occurrence. Categorical value scale; 1 = very bad, >50 abnormal, 2 = bad, >25% abnormal, 3 = average, 25% abnormal, 4 = good, <2% abnormal, and 5 = excellent, 100% normal. The vertical line separates baseline gourami from transported gourami.

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123 Figure 6-3. Gourami appearance as a percentage of occurrence of sellable versus unsellable tanks of fish ( N = 25 gourami observed for each treatment per observation time) from time 0 to 12 hours and 7 days post-transpor tation. Letters de note significantly different groupings ( P < 0.05) with standard error bars The vertical line separates baseline gourami from transported gourami.

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124 Figure 6-4. Gourami behavior scores ( N = 25 gourami observed for each metomidate treatment per observation time) from time 0 to 12 hours and 7 days post-transportation represented as a percentage of occurrence. Categorical scale; 1 = Very bad, >50% fish abnormal, 2 = Bad, >25% of fish abnormal, 3 = Average, 25% fish abnormal, and 4 = Excellent, 100% of fish normal. Letters denote significantly different groupings ( P < 0.05). The vertical line separates baseline gourami from transported gourami.

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125 Figure 6-5. Gourami behavior as a percentage of occurrence of sellable versus unsellable tanks of fish ( N = 25 gourami observed for each treatment per observation time) from time 0 to 12 hours and 7 days post-transportati on. Letters denote si gnificantly different groupings ( P < 0.05) with standard error bars. The vertical line separates baseline gourami from transported gourami.

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126 Figure 6-6. Gourami activity scores ( N = 25 gourami observed for each metomidate treatment per observation time) from time 0 to 12 hours and 7 days post-transportation represented as a percentage of occurrence. Categorical value scale; 1 =Not active, 2 = Less active, 3 = Active, 4 = More active, and 5 = Very active/Hyperactive. The vertical line separate s baseline gourami from transported gourami.

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127 CHAPTER 7 EXPERIMENT DISCUSSION Transportation of koi Cyprinus carpio and threespot gouram i Trichogaster trichopterus is stressful and may negatively impact the percentage of sellable fishes after transport. At time 0 hour post-transportation, both koi and threespot gourami transpor ted with 0 mg/L metomidate had increased plasma cortisol levels and blood gluc ose levels, respectively, compared to baseline fishes that were not transported, but not comp ared to other metomidate treated fishes. Additionally, 15% of the tanks of koi and 30% of the tanks of thr eespot gourami transported with 0 mg/L metomidate were cons idered unsellable immediately after transportation based on appearance and behavior. Clearly transportation is a significant stressor that affects the marketability of the fishes. The use of a sedative for transportation of fishes may reduce the fishes sensory perception and thereby diminish production of plasma cortisol and blood glucose. In teleost fishes, post-stress plasma cortisol levels typically range from 40 ng/mL (Pickering and Pottinger 1989), but in some species plasma cortisol levels of stressed fishes may reach as high as 1000 ng/mL (Barton and Iwama 1991). In this study, koi transported with 0 mg/L metomidate and sampled immediately after transportation ha d plasma cortisol levels that increased significantly from basal levels of 7.6.2 ng/mL to 236.3.1 ng/mL. The post-transport plasma cortisol levels are similar to previous ly reported levels for car p after acute stress. Pottinger (1998) and Carballo et al. (2005) bot h reported that common carp plasma cortisol levels were 240 ng/mL after exposure to 2 to 4 hours net confinement. Harms (1999) and Ross and Ross (1999) reported that in fishes exposed to metomidate, stage 3 anesthesia is induced without an elevation in plasma cortisol levels. Howeve r, in this study, a rise in koi

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128 plasma cortisol levels was inhibited in transported koi sedate d with 3.0 and 4.0 mg/L metomidate. With regards to the blood glucose results, as expected, blood glucose levels in baseline koi that were not transported ranged from 17 mg/dL which are similar to previously reported biochemical reference intervals for koi plasma le vels by Palmeiro et al. (2007) of 20 mg/dL. During transportation, the us e of metomidate to sedate koi di d not inhibit an elevation in blood glucose levels. Similarly, there was no inhibitio n of a rise in blood glucose levels in gourami transported with metomidate. But, there appe ared to be a downward trend in blood glucose levels for gourami transported with 0.2 and 0.3 mg /L metomidate. Possible explanations for the difference in significance obser ved between the blood glucose le vels of koi and threespot gourami was due to the concentratio ns of metomidate used, the dens ities in the shipping bags, or the species variability. The use of metomidate yielded varied result s for observations app earance and behavior with the species tested. For koi, there was a benefit to appearance when transported with 3.0 mg/L metomidate, but not with other concentra tions tested. Therefore, this concentration represented the best choice for transportation of this species in this study. However, for threespot gourami, there were no beneficial eff ects of metomidate use on either appearance or behavior. In fact, the use of 0.3 mg/L metomidate had a detrimen tal effect to gourami behavior, but there was no obvious benefit or detriment at the other metomidate concentrations tested. In summary, the effect of metomidate sedation during transportation on koi plasma cortisol and blood glucose levels and threespot gourami blood glucose levels was studied. In addition, the effect on koi and threespot gourami appearance, behavior, and activity level was studied as well. The results of this experiment indicate that koi plasma cortisol levels were not

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129 elevated and appearance was maintained with the use of metomidate at 3.0 mg/L during transportation In contrast, the use of metomidate in threespot gourami was not as clear. Metomidate concentrations of 0.1, 0.2, and 0.3 mg /L resulted in a non-si gnificant, non-linear inhibition of blood glucose level rise, but there were no obvious benefici al effects on appearance and behavior. Additional studies with threespo t gourami should be conducted to determine the advantage, if any, of metomidate sedation during transportation for this species. In conclusion, this study demonstrates the use of metomidate se dation during transportatio n of koi and threespot gourami minimized an increase in blood chemistry parameters, traditionally indicative of the stress response. Therefore, the use of metomi date may be a valuable aid for researchers to inhibit a rise in plasma cortisol and blood glucose levels in res ponse to acute stress.

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130 APPENDIX A APPEARANCE, BEHAVIOR, AND ACTIVIT Y EVALUATION FORM A. Fish Appearance (percentage based on general impression, no need to do strict counts) 1BadUnsellableMore than 50% of the fish abnormal with minor to moderate damage of body/fins/eyes 2BadUnsellable% of the fish abnorma l with minor to moderate damage of body/fins/eyes OR greater than 10% of fish a bnormal with moderate to severe damage of body/fins/eyes 3AverageLess than 25% of fish abnormal, fin/body/eye damage minor 4GoodLess than 2% abnormal, fish overall normal, very little abnormalities noted 5Excellent% of fish normal, overall extremely healthy looking What is the overall evaluation of condition/appearance? 1 2 3 4 5 Considerations for evaluating condition/appear ance (consider severity of abnormality also) Body Fins Eyes Other General_ Coloration Ragged/Torn Popeye (Exophthalmia) Other lesions Dropsy (bloated) Clamped Cloudy Parasites Hemorrhaging Hemorrhaging Hemorrhaging Scale loss Deformities Deformities Deformities B. Fish Behavior (percentage based on general impression, no n eed to do strict counts) 1Very BadUnsellableMore than 50% of the fish abnormal 2BadUnsellable% of the fish abnormal 3AverageLess than 25% of the fish abnormal 4Excellent% fish be having normally, alert What is the overall evaluation of behavior ? 1 2 3 4 Considerations for evaluating abnormal behavior Breathing/Respiration S wimming General Piping (sucking in air at top) Spinning Isolated/Hiding Gasping (wide flaring of gill plate) Flashing (scratching) Loss of Located in an buoyancy abnormal part of C. Activity Level water column___________________________ 1 Not active 2 Less activity 3 Active 4 More active 5 Very Active/Hyperactive What is the overall evaluation of activity level? 1 2 3 4 5__ Figure A-1. Appearance, behavior and activity level evaluation fo rm used to evaluate a score for tanks of fish post-transportation as indicators of marketability.

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131 APPENDIX B KOI DATA

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132Table B-1. Mean (SD) koi plasma cortisol levels in ng/mL. Different letters indi cate significant differences down columns (P < 0.05). Metomidate Time 0 hour a Time 2 hours b Time 6 hours cTime 12 hours d Baseline 34.8 28.5 z 32.1 34.1 z117.7 58.4165.0 50.2 0 mg/L 310.7 28.5 x 200.6 34.1 y265.6 58.4162.6 64.8 1.0 mg/L 238.3 28.5 xy 115.8 39.4 yz225.2 52.3135.3 67.8 2.0 mg/L 219.4 25.5 xy 214.0 30.5 y215.8 67.5306.3 56.1 3.0 mg/L 125.0 25.5 yz 237.0 30.5 y145.3 52.3192.5 50.2 4.0 mg/L 139.3 28.5 yz 248.1 34.1 y256.1 67.5124.6 56.1 a ANOVA: F = 10.75; df = 5, 19; P < 0.0001. b ANOVA: F = 7.03; df = 5, 20; P = 0.0006. c ANOVA: F = 0.85; df = 5, 16; P = 0.54. d ANOVA: F = 1.34; df = 5, 18; P = 0.29. Table B-2. Mean (SD) koi blood glucose levels in mg/dL. Different lett ers indicate signifi cant differences down columns (P < 0.05). Metomidate Time 0 hour a Time 2 hours b Time 6 hours c Time 12 hours d Baseline 54.9 19.4 31.1 13.2 z39.8 52.135.3 8.5 0 mg/L 25.3 17.3 68.2 13.2 yz72.8 45.1 67.0 10.9 1.0 mg/L 90.2 19.4 107.2 14.7 y85.2 45.174.6 9.5 2.0 mg/L 52.7 19.4 83.3 13.2 yz199.1 45.159.0 9.5 3.0 mg/L 45.3 17.3 67.3 13.2 yz80.3 40.455.7 8.5 4.0 mg/L 65.8 17.3 100.2 13.2 y76.9 45.160.5 8.5 a ANOVA: F = 1.39; df = 5, 21; P = 0.27. b ANOVA: F = 4.10; df = 5, 23; P = 0.008. c ANOVA: F = 1.40; df = 5, 18; P = 0.27. d ANOVA: F = 2.22; df = 5, 20; P = 0.09.

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133Table B-3. Mean (SD) koi appearance sc ores. Different letters i ndicate significant differenc es across rows (P < 0.05). Time Baseline 0 mg/L a1.0 mg/L2.0 mg/L3.0 mg/L4.0 mg/L 0 hour b 86.0 17.1 z 43.2 21.8 y70.6 5.6 yz53.4 21.5 yz64.5 26.1 yz40.1 14.0 y 1 hour c 72.7 16.7 52.0 12.467.6 11.348.6 18.467.0 13.145.3 12.6 4 hours d 113.3 33.2 59.8 36.375.7 11.961.6 24.357.4 36.757.4 30.7 8 hours e 81.6 18.2 53.4 22.166.1 9.949.8 18.761.6 11.155.0 12.6 12 hours f 73.6 23.1 49.3 22.166.9 22.050.6 18.766.2 13.056.4 12.0 7 days g 63.6 .9 49.3 19.367.9 .759.3 26.062.3 14.160.8 .1 a All treatments are metomidate concentrations in mg/L. b KW: H = 12.5; df = 5, 24; P = 0.03. c KW: H = 12.02; df = 5, 24; P = 0.03, Even though Kruskal-Wallis indicates significance, Dunns comparison does not. d KW: H = 9.41; df = 5, 24; P = 0.09. e KW: H = 9.74; df = 5, 24; P = 0.08. f KW: H = 5.29; df = 5, 24; P = 0.38. g KW: H = 3.19; df = 5, 24; P = 0.67. Table B-4. Mean (SD) per centage of sellable tanks of koi based on appearance. Time Baseline 0 mg/L a1.0 mg/L2.0 mg/L3.0 mg/L4.0 mg/L 0 hour b 1.00 0.85 0.14 0.95 0.110.85 0.220.85 0.220.75 0.18 1 hour c 0.95 0.11 0.90 0.141.000.85 0.220.95 0.110.90 0.14 4 hours d 1.00 0.95 0.111.000.95 0.110.95 0.110.95 0.11 8 hours e 1.00 0.90 0.141.000.95 01.001.00 12 hours f 1.00 0.95 0.111.000.90 0.220.95 0.111.00 7 days g 1.00 1.001.001.001.001.00 a All treatments are metomidate concentrations in mg/L. b ANOVA: F = 1.65; df = 5, 24; P = 0.19. c ANOVA: F = 0.67; df = 5, 24; P = 0.65. d ANOVA: F = 0.40; df = 5, 24; P = 0.84. e ANOVA: F = 1.68; df = 5, 24; P = 0.18. f ANOVA: F = 0.62; df = 5, 24; P = 0.68. g No differences among treatments; ther efore, no statistica l test was run.

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134Table B-5. Mean (SD ) koi behavior scores. Time Baseline 0 mg/L a1.0 mg/L2.0 mg/L3.0 mg/L4.0 mg/L 0 hour b 64.4 13.6 58.9 13.450.6 7.861.5 15.167.0 11.560.6 6.0 1 hour c 68.8 6.6 59.8 12.865.8 8.350.8 8.353.6 19.565.7 8.2 4 hours d 61.6 5.0 67.4 5.056.9 5.064.5 5.065.6 5.047.1 5.0 8 hours e 59.0 4.0 62.0 4.062.0 4.056.0 4.059.0 4.059.0 4.0 12 hours f 59.1 5.6 52.8 5.659.1 5.662.1 5.665.0 5.665.0 5.6 7 days g 61.1 3.8 55.8 3.861.7 .861.7 3.864.6 3.858.7 .8 a All treatments are metomidate concentrations in mg/L. b KW: H = 6.23; df = 5, 24; P = 0.28. c KW: H = 8.5; df = 5, 24; P = 0.13. d KW: H = 8.9; df = 5, 24; P = 0.12. e KW: H = 1.6; df = 5, 24; P = 0.90. f KW: H = 3.06; df = 5, 24; P = 0.69. g KW: H = 2.91; df = 5, 24; P = 0.71. Table B-6. Mean (SD) per centage of sellable tanks of koi based on behavior. Time Baseline 0 mg/L a1.0 mg/L2.0 mg/L3.0 mg/L4.0 mg/L 0 hour b 0.90 0.14 0.85 0.140.85 0.141.001.000.95 0.11 1 hour c 1.00 1.001.001.001.001.00 4 hours d 0.95 0.11 0.95 0.111.000.95 0.110.95 0.111.00 8 hours e 1.00 1.001.001.001.001.00 12 hours f 1.00 0.95 0.111.001.001.001.00 7 days g 0.95 0.11 1.001.001.001.000.95 0.11 a All treatments are metomidate concentrations in mg/L. b ANOVA: F = 2.07; df = 5, 24; P = 0.10. c No differences among treatments; therefore, no statistical test was run. d ANOVA: F = 0.40; df = 5, 24; P = 0.84. e No differences among tr eatments; therefore, no statistical test was run. f ANOVA: F = 1.00; df = 5, 24; P = 0.44. g ANOVA: F = 0.80; df = 5, 24; P = 0.56.

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135Table B-7. Mean (SD) koi activity level scores. Different letters indicate sign ificant differences across rows (P < 0.05). Time Baseline 0 mg/L a1.0 mg/L2.0 mg/L3.0 mg/L4.0 mg/L 0 hour b 49.2 11.9 z 51.6 17.8 yz51.7 13.1 yz68.3 13.5 yz63.6 9.9 yz78.3 7.3 y 1 hour c 68.8 5.7 59.8 6.965.8 13.450.8 16.353.6 10.465.7 14.2 4 hours d 54.2 10.1 61.9 3.260.5 2.461.0 2.058.8 9.366.9 8.9 8 hours e 46.3 26.1 56.0 22.968.6 19.575.7 21.259.0 14.557.5 11.6 12 hours f 58.4 27.5 50.4 18.559.4 12.562.2 11.762.2 18.359.4 7.8 7 days g 56.8 17.2 61.7 17.067.8 11.059.6 20.764.4 20.447.9 12.0 a All treatments are metomidate concentrations in mg/L. b KW: H = 14.24; df = 5, 24; P = 0.01. c KW: H = 9.04; df = 5, 24; P = 0.11. d KW: H = 3.80; df = 5, 24; P = 0.58. e KW: H = 5.55; df = 5, 24; P = 0.35. f KW: H = 1.04; df = 5, 24; P = 0.96. g KW: H = 4.74; df = 5, 24; P = 0.45.

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136 APPENDIX C GOURAMI DATA

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137Table C-1. Mean (SD) gourami blood glucose levels in mg/dL. Different letters indicate significant differences down columns (P < 0.05). Metomidate Time 0 hour a Time 2 hours b Time 6 hours cTime 12 hours d Baseline 18.2 8.9 z 13.4 10.5 z23.7 21.422.0 9.0 z 0 mg/L 150.3 75.4 xy 98.1 45.1 y46.0 21.442.7 9.0 xyz 0.1 mg/L 124.6 52.2 xyz 146.4 40.2 y111.0 21.451.0 9.9 xy 0.2 mg/L 94.7 19.0 yz 101.6 48.1 y57.4 21.429.2 9.0 yz 0.3 mg/L 121.6 68.0 xyz 122.2 35.0 y82.6 21.452.2 9.9 xy 0.4 mg/L 225.1 174.0 x 217.9 121.2 x93.7 23.466.3 9.0 x a ANOVA: F = 2.98; df = 5, 25; P = 0.03. b ANOVA: F = 7.33; df = 5, 30; P = 0.0001. c ANOVA: F = 2.27; df = 5, 29; P = 0.07. d ANOVA: F = 3.21; df = 5, 28; P = 0.02. Table C-2. Mean (SD) gour ami appearance scores. Time Baseline 0 mg/L a 0.1 mg/L0.2 mg/L0.3 mg/L0.4 mg/L 0 hour b 16.5 4.1 18.4 2.9 17.5 3.415.9 2.117.6 1.617.7 0.8 2 hours c 18.7 5.7 20.1 3.0 18.6 3.221.1 2.220.2 3.321.6 2.0 4 hours d 16.4 5.1 17.3 2.4 15.4 1.418.0 1.319.3 3.019.2 2.1 6 hours e 15.4 4.8 19.8 4.4 21.5 1.419.3 2.919.0 3.016.2 3.7 12 hours f 16.1 4.4 18.8 3.9 18.5 3.120.3 5.121.0 5.118.5 4.4 7 days g 17.9 1.5 19.0 3.1 19.2 2.318.3 2.816.7 1.216.9 5.3 a All treatments are metomidate concentrations in mg/L. b KW: H = 3.37; df = 5, 24; P = 0.64. c KW: H = 3.68; df = 5, 24; P = 0.60. d KW: H = 6.16; df = 5, 24; P = 0.29. e KW: H = 9.05; df = 5, 24; P = 0.11. f KW: H = 3.58; df = 5, 24; P = 0.61. g KW: H = 3.50; df = 5, 24; P = 0.62.

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138Table C-3. Mean (SD) percenta ge of sellable tanks of gourami based on appearan ce. Different letters indicate significant dif ferences across rows (P < 0.05). Time Baseline 0 mg/L a1.0 mg/L2.0 mg/L3.0 mg/L4.0 mg/L 0 hour b 1.00 0.70 0.210.80 0.270.75 0.180.85 0.140.75 0.18 2 hours c 1.00 0.75 0.180.75 0.250.90 0.140.70 0.210.85 0.14 4 hours d 1.00 0.80 0.210.80 0.210.90 0.140.80 0.210.70 0.21 6 hours e 1.00 z 0.65 0.22 y0.90 0.14 yz0.85 0.14 yz0.80 0.21 yz1.00 y 12 hours f 1.00 z 0.55 0.21 y0.75 0.18 yz0.85 0.14 yz0.85 0.14 yz0.80 0.11 yz 7 days g 0.90 0.14 0.95 0.110.95 0.110.90 0.140.75 00.90 0.14 a All treatments are metomidate concentrations in mg/L. b ANOVA: F = 2.07; df = 5, 24; P = 0.10. c ANOVA: F = 2.23; df = 5, 24; P = 0.08. d ANOVA: F = 1.74; df = 5, 24; P = 0.16. e ANOVA: F = 4.39; df = 5, 24; P = 0.006. f ANOVA: F = 4.93; df = 5, 24; P = 0.003. g ANOVA: F = 2.00; df = 5, 24; P = 0.12. Table C-4. Mean (SD) gourami behavior sc ores. Different letters i ndicate significant differenc es across rows (P < 0.05). Time Baseline 0 mg/L a0.1 mg/L0.2 mg/L0.3 mg/L 0.4 mg/L 0 hour b 22.6 0.4 z 17.0 3.2 yz16.3 4.0 yz18.4 3.8 yz15.5 4.0 y 19.2 4.2 yz 2 hours c 25.5 2.6 19.9 3.119.2 4.022.9 3.117.4 6.8 21.5 5.2 4 hours d 30.0 5.4 25.1 6.421.5 6.427.1 3.820.7 6.0 29.1 2.2 6 hours e 18.4 2.2 16.7 3.916.2 2.417.5 2.115.9 2.8 20.1 1.8 12 hours f 15.7 2.0 15.0 3.416.0 2.718.5 1.717.4 2.7 18.3 0.9 7 days g 44.3 5.7 54.5 0.052.0 5.749.3 7.244.3 10.7 52.0 5.7 a All treatments are metomidate concentrations in mg/L. b KW: H = 13.68; df = 5, 24; P = 0.02. c KW: H = 8.93; df = 5, 24; P = 0.11. d KW: H = 9.75; df = 5, 24; P = 0.08. e KW: H = 8.49; df = 5, 24; P = 0.13. f KW: H = 8.28; df = 5, 24; P = 0.14. g KW: H = 9.11; df = 5, 24; P = 0.10.

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139Table C-5. Mean (SD) percenta ge of sellable tanks of gourami based on behavior. Different letters indicate significant diffe rences across rows (P < 0.05). Time Baseline 0 mg/L a1.0 mg/L2.0 mg/L3.0 mg/L4.0 mg/L 0 hour b 1.00 z 0.55 0.33 yz0.85 0.14 yz0.70 0.27 yz0.50 0.25 y0.75 0.35 yz 2 hours c 0.90 0.14 0.80 0.210.75 0.180.85 0.140.60 0.290.80 0.21 4 hours d 0.85 0.14 0.85 0.140.70 0.110.85 0.140.80 0.110.95 0.11 6 hours e 0.75 0.18 0.60 0.380.65 0.290.70 0.210.55 0.270.85 0.14 12 hours f 0.60 0.29 0.40 0.420.55 0.330.75 0.180.65 0.290.85 0.14 7 days g 0.90 0.14 1.001.000.90 0.140.90 0.141.00 a All treatments are metomidate concentrations in mg/L. b ANOVA: F = 3.23; df = 5, 24; P = 0.02. c ANOVA: F = 0.95; df = 5, 24; P = 0.47. d ANOVA: F = 1.93; df = 5, 24; P = 0.13. e ANOVA: F = 0.82; df = 5, 24; P = 0.55. f ANOVA: F = 1.22; df = 5, 24; P = 0.33. g ANOVA: F = 1.60; df = 5, 24; P = 0.20. Table C-6. Mean (SD) gour ami activity level scores. Time Baseline 0 mg/L a 0.1 mg/L0.2 mg/L0.3 mg/L0.4 mg/L 0 hour b 25.8 7.9 25.9 5.0 30.0 5.324.0 6.323.9 3.428.1 3.9 2 hours c 24.3 5.0 22.8 4.7 23.4 7.022.3 6.923.4 4.926.3 2.5 4 hours d 27.6 1.9 22.2 4.4 22.2 4.924.3 5.423.4 5.223.1 4.1 6 hours e 26.7 1.6 28.6 3.4 25.2 3.628.2 2.427.1 2.926.3 2.7 12 hours f 25.8 2.4 21.0 8.0 23.9 4.525.4 2.824.6 3.725.8 2.1 7 days g 39.4 4.2 38.4 5.7 38.4 5.738.4 0.036.3 4.840.0 3.6 a All treatments are metomidate concentrations in mg/L. b KW: H = 4.35; df = 5, 24; P = 0.50. c KW: H = 1.57; df = 5, 24; P = 0.90. d KW: H = 5.46; df = 5, 24; P = 0.36. e KW: H = 4.03; df = 5, 24; P = 0.55. f KW: H = 2.63; df = 5, 24; P = 0.76. g KW: H = 1.63; df = 5, 24; P = 0.90.

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152 BIOGRAPHICAL SKETCH Tina Christine Crosby was born in S pringfield, Illinois; but grew up primarily in southern California and throughout south and central Flor ida. After she graduated high school, Tina attended Palm Beach Community College for two year s. Tina received her Bachelor of Science degree in biology from the University of South Florida in Tampa, Florida. After graduation, Tina worked as a research biologist for th e University of Florida Tropical Aquaculture Laboratory (TAL) in Ruskin, Florida on a gr ant to improve the harvesting, grading, and transportation of ornamental fishes. While at TAL, Tina completed an Associate of Science degree in aquaculture at Hillsborough Community Colle ge in Tampa, Florida. After 2 years at TAL, Tina began a Master of Science program th at focused on fish health management at the University of Florida, School of Forest Resources and Conservation, Program in Fisheries and Aquatic Sciences. Tina has accepted a position w ith the University of Florida, College of Veterinary Medicine as Dr. Denise Pettys biological scientist in the fish health diagnostic laboratory.