Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Assessment of distress associated with carbon dioxide euthanasia in laboratory rats Niel, Lee Erin 2006

You don't seem to have a PDF reader installed, try download the pdf

Item Metadata

Download

Media
[if-you-see-this-DO-NOT-CLICK]
ubc_2006-200599.pdf [ 6.53MB ]
Metadata
JSON: 1.0093022.json
JSON-LD: 1.0093022+ld.json
RDF/XML (Pretty): 1.0093022.xml
RDF/JSON: 1.0093022+rdf.json
Turtle: 1.0093022+rdf-turtle.txt
N-Triples: 1.0093022+rdf-ntriples.txt
Original Record: 1.0093022 +original-record.json
Full Text
1.0093022.txt
Citation
1.0093022.ris

Full Text

ASSESSMENT OF DISTRESS ASSOCIATED WITH CARBON DIOXIDE EUTHANASIA IN LABORATORY RATS by LEE ERIN NIEL B.Sc, Simon Fraser University, 2000  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES  (Animal Science)  The University of British Columbia July 2006 © Lee Erin Niel, 2006  Abstract C a r b o n d i o x i d e (CO2) gas is the most w i d e l y u s e d euthanasia agent for  laboratory  rodents. H o w e v e r , it has the potential to cause b o t h p a i n and d y s p n e a , an unpleasant sensation o f breathless, w h i l e a n i m a l s are still c o n s c i o u s . T h e a i m s o f this dissertation were to determine whether g r a d u a l - f i l l CO2 euthanasia causes distress i n laboratory rats, and to e x a m i n e potential sources  o f distress, i n c l u d i n g p a i n , d y s p n e a a n d n o v e l t y . o f rats d u r i n g g r a d u a l - f i l l CO2  The  first  study  examined  euthanasia. R a t s s h o w e d  the  behavioural  responses  increased  exploratory  b e h a v i o u r s and escape b e h a v i o u r s d u r i n g CO2 euthanasia, suggesting that this  procedure does cause distress. T h e second and t h i r d studies u s e d a p p r o a c h - a v o i d a n c e testing to investigate a v e r s i o n to CO2 i n rats, b y e x a m i n i n g their w i l l i n g n e s s to enter a test cage c o n t a i n i n g CO2 for access to an attractive f o o d r e w a r d . R a t s were f o u n d to a v o i d CO2 concentrations that are s u f f i c i e n t to cause u n c o n s c i o u s n e s s . S p e c i f i c a l l y , w h e n tested w i t h static concentrations o f CO2 rats s h o w e d a v o i d a n c e at 1 5 % CO2 a n d greater, a n d w h e n tested w i t h g r a d u a l l y i n c r e a s i n g concentrations o f CO2 at f l o w rates r a n g i n g f r o m 3 to 2 7 % per m i n u t e , rats s h o w e d a v o i d a n c e at 13 to 1 6 % CO2. T h i s a v o i d a n c e indicates that rats are at least m o d e r a t e l y averse to  CO2  concentrations o c c u r r i n g d u r i n g g r a d u a l - f i l l CO2 euthanasia, and that f o r c e d exposure l i k e l y causes distress. C o n c e n t r a t i o n s o f CO2 that were associated w i t h b e h a v i o u r a l responses and aversion were  not consistent w i t h p r e v i o u s  data o n p a i n due to CO2. H o w e v e r ,  similar  concentrations have been s h o w n to cause d y s p n e a i n h u m a n s . T h e final study e x a m i n e d the role o f n o v e l t y i n rats' responses to CO2, and f o u n d that n o v e l t y w a s not a m a j o r source o f distress d u r i n g g r a d u a l - f i l l CO2 euthanasia. In s u m m a r y , these studies suggest that g r a d u a l - f i l l CO2 euthanasia causes distress i n rats, and that this distress is l i k e l y due to d y s p n e a . Further research is necessary to e x a m i n e the effects o f CO2 o n other rodent species s u c h as m i c e , and to i d e n t i f y alternative methods o f euthanasia that cause u n c o n s c i o u s n e s s w i t h o u t distress.  Table of Contents Abstract  ii  Table o f Contents  iii  List o f Tables  v  List o f Figures  vi  List of Abbreviations  vii  Acknowledgements  viii  C o - A u t h o r s h i p Statement  ix  C H A P T E R 1: G e n e r a l Introduction  1  1.1 Introduction  1  1.2 C a r b o n D i o x i d e E u t h a n a s i a  3  1.3 A n i m a l Distress and C a r b o n D i o x i d e  9  1.4 A s s e s s m e n t o f Distress D u r i n g CO2 E u t h a n a s i a 1.5 O b j e c t i v e s  •.  16 27  1.6 R e f e r e n c e s  29  C H A P T E R 2 : B e h a v i o u r a l responses o f rats to g r a d u a l - f i l l c a r b o n d i o x i d e euthanasia and r e d u c e d o x y g e n concentrations  41  2.1 I n t r o d u c t i o n  41  2.2 M a t e r i a l s and M e t h o d s  43  2.3 R e s u l t s  47  2.4 D i s c u s s i o n  49  2.5 R e f e r e n c e s  61  C H A P T E R 3 : R a t s a v o i d exposure to c a r b o n d i o x i d e and a r g o n . . . . 3.1 Introduction  66 66  3.2 M a t e r i a l s and M e t h o d s  67  3.3 R e s u l t s  71  3.4 D i s c u s s i o n  73  3.5 R e f e r e n c e s  81  C H A P T E R 4: E f f e c t o f f l o w rate o n a v e r s i o n to g r a d u a l - f i l l c a r b o n d i o x i d e euthanasia i n rats 85 4.1 Introduction  85  4.2 M a t e r i a l s and M e t h o d s  87  4.3 R e s u l t s  90  4.4 D i s c u s s i o n  91  4.5 R e f e r e n c e s  95  C H A P T E R 5 : E f f e c t s o f n o v e l t y o n rat responses to CO2 exposure 5.1 I n t r o d u c t i o n  97 97  5.2 M a t e r i a l s and M e t h o d s 5.3 R e s u l t s ,  99 104  iii  5.4 D i s c u s s i o n  106  5.5 R e f e r e n c e s  113  C H A P T E R 6: G e n e r a l D i s c u s s i o n  116  6.1 D i s t r e s s i n rats d u r i n g CO2 euthanasia  116  6.2 Sources o f distress  117  6.3 C r i t i q u e o f methods  122  6.3 Future directions  125  6.4 C o n c l u s i o n s  129  6.5 R e f e r e n c e s  130  iv  List of Tables Table 2.1  Descriptions  o f rat b e h a v i o u r s  recorded  during  baseline  and during  exposure to C O 2 or r e d u c e d O2 concentrations  Table 2.2  56  D i f f e r e n c e f r o m baseline f o r each o f the f i v e b e h a v i o u r a l responses o f rats d u r i n g air a n d C O 2 exposure (n =' 8 rats). D a t a are presented as m e d i a n s with 25  Table 2.3  Table 3.1  t h  and 7 5  t h  percentiles, a n d statistical c o m p a r i s o n s were m a d e w i t h  W i l c o x o n S i g n e d R a n k s Test (T; based o n N values >0)  57  D i f f e r e n c e f r o m baseline f o r each o f the f i v e b e h a v i o u r a l responses o f rats d u r i n g air a n d exposure to r e d u c e d O2 concentrations (n = 8 rats). D a t a are presented as m e d i a n s w i t h 2 5 t h a n d 7 5 t h percentiles, a n d statistical c o m p a r i s o n s w e r e m a d e w i t h the W i l c o x o n S i g n e d R a n k s Test ( T ; based o n N values>0)  58  M e d i a n (with 2 5  t h  and 7 5  t h  percentiles) n u m b e r o f r e w a r d items eaten  d u r i n g the entire s e s s i o n a n d eating a n d d w e l l i n g t i m e s d u r i n g the first entry w i t h either air o r argon i n the test cage (n = 9 rats) Table 5.1  Descriptions  o f rat b e h a v i o u r s  recorded  during  baseline  exposure to C O 2 o r p e p p e r m i n t odour ( E x p e r i m e n t 3)  v  79 and during 110  List of Figures Figure 2.1  A v e r a g e concentrations o f O2 (open m a r k e r s ) a n d CO2 ( f i l l e d m a r k e r s ) i n the c h a m b e r d u r i n g the first 6 0 0 s o f the f i l l i n g process. C o n c e n t r a t i o n s were t a k e n 5 c m (triangles)  a n d 15 c m (squares) f r o m the c h a m b e r  bottom  Figure 2.2  59  R e s p o n s e s b y rats d u r i n g the baseline p e r i o d and t h e n d u r i n g exposure to either air ( f i l l e d squares) or CO2 (open squares) starting at t = 0. M e d i a n values per 1 5 s p e r i o d are s h o w n for a) a c t i v i t y , b) rears , c) nose to l i d , d) escape b e h a v i o u r s , and e) v o c a l i z a t i o n s (n = 8 rats)  Figure 3.1  .60  M e d i a n ( ± interquartile ranges) a) n u m b e r o f r e w a r d items eaten, b) eating t i m e ( f i l l e d squares) and d w e l l i n g t i m e ( o p e n squares) for the first entry, and c) n u m b e r o f attempted entries into the test cage d u r i n g sessions w i t h 0, 5 , 10, 15 a n d 2 0 % C 0  Figure 4.1  2  .80  (n = 9 rats)  L e a s t squares m e a n ( ± S E M ) a) n u m b e r o f r e w a r d items eaten, b) latency to stop eating (open) a n d to leave the test cage ( f i l l e d ) , a n d c)  CO2  c o n c e n t r a t i o n at the t i m e w h e n rats stopped eating (open) a n d left the test cage (filled) d u r i n g test sessions w i t h CO2 flow rates o f 3 , 7, 14, 2 0 and .94  2 7 % o f the test cage v o l u m e per m i n u t e (n = 8 rats)  Figure 5.1  A p p r o a c h - a v o i d a n c e responses to CO2 for the first three days and for the final  day (Day  16) o f exposure ( E x p e r i m e n t  1). M e a n ( ± S E M )  latency to stop eating a n d leave the test cage, (b) C 0  2  (a)  concentration w h e n  rats stopped eating a n d left the test cage, a n d (c) n u m b e r o f r e w a r d items eaten (n - 9 rats)  Figure 5.2  Ill  B e h a v i o u r a l responses o f rats to CO2 euthanasia a n d p e p p e r m i n t odour exposure ( E x p e r i m e n t 3). L e a s t squares m e a n ( ± S E M ) (a) n u m b e r of rears, and (b) t i m e spent w i t h the nose i n contact w i t h the test cage l i d d u r i n g baseline a n d d u r i n g exposure to either C 0 w i t h p e p p e r m i n t odour (n = 16 rats)  VI  2  (n = 16 rats) or air 112  List of Abbreviations ACTH  adrenocorticotropic h o r m o n e  CNS  C e n t r a l n e r v o u s system  co  Carbon dioxide  2  CRH  Corticotrophin-releasing hormone  CSF  Cerebral spinal fluid  EEG  Electroencephalogram  HPA  H y p o t h a l a m i c - p i t u i t a r y - a d r e n a l axis  o  Oxygen  2  SAM '  S y m p a t h e t i c - a d r e n e r g i c - m e d u l l a r y axis  SEP  S o m a t o s e n s o r y - e v o k e d potential  USV  Ultrasonic vocalization  vii  Acknowledgements I am  grateful  encouragement.  to  my  supervisor,  Dan Weary, for his  Dan has allowed me tremendous  endless  enthusiasm  and  freedom to pursue research that I am  passionate about. He has also given me every opportunity to learn about all aspects of academia, from writing grants to teaching and training students. I am especially thankful for his constant barrage of questions and criticisms, which have been crucial to my growth as an independent and critical researcher. I have thoroughly enjoyed my disagreements with Dan, and I hope that future colleagues are as forthcoming with their opinions. I would like to thank my committee members, David Fraser, Jim Love and K e n Craig for their insight into my thesis area, and for their help with editing. Dave has been a fount o f knowledge on both animal welfare and grammar, and I have learned vast amounts from him during my time in the Animal Welfare Program. I am grateful to Jim and K e n for their scepticism, which has made me think carefully about appropriate interpretation and presentation of my data.  '  I would also like to thank a number of people who have made contributions to the studies in this thesis. First and foremost, I want to thank Cassandra Tucker, with whom I have spent countless hours discussing research ideas, experimental design and statistical analysis. I would also like to thank Richard Kirkden, for his insights on animal motivation. Gilles Galzi, Sylvia Leung and Jurgen Pehlke have been incredibly helpful with the day to day running of the lab. Murray Hodgson provided technical knowledge on ultrasound, and very generously lent me equipment for the ultrasound recordings in Chapter 2. Sarah Stewart, Marianne Pfaffinger and Gary Lee provided assistance with day to day lab duties and with data collection (Sarah, Chapters 4 and 5; Marianne, Chapters 3 and 5; Gary, Chapter 5). Mitja Sedlbauer, Nicole Fenwick, and Amanda Grout provided assistance with animal care. I am also thankful to the many members of the Animal Welfare Program for their support and friendship. Lunchtime discussions and presentations have been a high point in my time as a graduate student. I am eternally indebted to my family and friends for their unwavering support. A special thank you goes to my husband, Colin, who has helped me to stay sane throughout this process and has done far more than his fair share of the dog-walking these past few months. Finally, completion of this work would not have been possible without the generous financial support that I received through the National Sciences and Engineering Research Council and the U B C Faculty o f Land and Food Systems (Elizabeth Roxann Howland Fellowship, Leonard S. Klink Memorial Fellowship, Beryl March Travel Award and Charles River Laboratories Scholarship).  viii  Co-Authorship Statement Chapter 2: I w a s r e s p o n s i b l e for identifying the r e s e a r c h a r e a , e x p e r i m e n t a l d e s i g n , d a t a collection, statistical a n a l y s e s , a n d writing t h e m a n u s c r i p t . D a n W e a r y contributed to t h e e x p e r i m e n t a l d e s i g n a n d to the interpretation a n d presentation of the m a n u s c r i p t .  Chapter 3: I w a s r e s p o n s i b l e for identifying the r e s e a r c h a r e a , e x p e r i m e n t a l d e s i g n , d a t a collection, statistical a n a l y s e s , a n d writing the manuscript. D a n W e a r y contributed to the e x p e r i m e n t a l d e s i g n a n d to t h e interpretation a n d presentation of the m a n u s c r i p t .  Chapter 4: I w a s r e s p o n s i b l e for a portion of the d a t a collection, a n d for identifying the r e s e a r c h a r e a , e x p e r i m e n t a l d e s i g n , statistical a n a l y s e s , a n d writing the m a n u s c r i p t . S a r a h Stewart contributed to t h e e x p e r i m e n t a l d e s i g n a n d data collection. D a n W e a r y contributed to the e x p e r i m e n t a l d e s i g n a n d to the interpretation a n d presentation of the m a n u s c r i p t .  Chapter 5: I w a s r e s p o n s i b l e for identifying the r e s e a r c h a r e a , e x p e r i m e n t a l d e s i g n , d a t a collection, statistical a n a l y s e s , a n d writing the manuscript. D a n W e a r y contributed to t h e e x p e r i m e n t a l d e s i g n , a n d to the interpretation a n d presentation of the m a n u s c r i p t .  ix  CHAPTER 1: General Introduction 1.1 Introduction In  the i n d u s t r i a l i z e d countries,  a n i m a l s are r o u t i n e l y  bred  and used i n  e x p e r i m e n t a t i o n that serves to advance b a s i c b i o l o g i c a l k n o w l e d g e ,  scientific  advance h u m a n  veterinary m e d i c i n e , a n d ensure the safety o f h u m a n s , a n i m a l s and the e n v i r o n m e n t  and  through  regulatory testing. R o d e n t s are the m o s t w i d e l y u s e d species i n this research, and i n C a n a d a alone a p p r o x i m a t e l y one m i l l i o n m i c e a n d rats are used i n research each year ( C C A C , 2 0 0 6 ) T h e vast m a j o r i t y o f research and b r e e d i n g a n i m a l s are e v e n t u a l l y k i l l e d , either for e x p e r i m e n t a l purposes or to reduce surplus stock. D u r i n g an e x p e r i m e n t , a n i m a l s m i g h t be k i l l e d i n order to alleviate p a i n and s u f f e r i n g due to a n e x p e r i m e n t a l m a n i p u l a t i o n or to a l l o w for tissue c o l l e c t i o n and analysis. T h e t e r m euthanasia is c o m m o n l y used to refer to the k i l l i n g o f research a n i m a l s . E u t h a n a s i a is d e r i v e d f r o m G r e e k for ' g o o d d e a t h ' , w h i c h suggests a process w h i c h does not i n v o l v e p a i n or distress ( B l a c k m o r e , 1993). A l t h o u g h i n practice it m a y not be p o s s i b l e to d e v e l o p a procedure for the k i l l i n g o f a n i m a l s that is c o m p l e t e l y d e v o i d o f stress, the g o a l is c l e a r l y to m i n i m i z e any p a i n and distress associated w i t h the procedure. A n u m b e r o f different euthanasia methods are currently a p p r o v e d for k i l l i n g laboratory rodents i n C a n a d a ( C C A C , 1993). E u t h a n a s i a g u i d e l i n e s d e v e l o p e d b y the C a n a d i a n C o u n c i l o n A n i m a l C a r e are generally i n l i n e w i t h the suggested euthanasia methods o f other  western  countries s u c h as those o f the U S A ( A V M A , 2 0 0 1 ) , the U K ( U K H o m e O f f i c e , 1997), countries o f the E u r o p e a n U n i o n ( C l o s e et a l . , 1997) and A u s t r a l i a a n d N e w Z e a l a n d ( A N Z C C A R T , 1993). T h e s e methods i n c l u d e p h y s i c a l techniques, injectable anaesthetics, and anaesthetic and non-anaesthetic gases. It is r e c o g n i z e d that s o m e o f these methods have the potential to result i n b o t h p a i n and distress, but the c h o s e n m e t h o d does not depend s o l e l y o n a r e d u c t i o n o f a n i m a l  1  s u f f e r i n g . It also depends o n p r a g m a t i c concerns s u c h as the purpose o f the k i l l i n g and the constraints o f t i m e , m o n e y , and safety to h u m a n s , other a n i m a l s a n d the e n v i r o n m e n t . T h e C a n a d i a n C o u n c i l o n A n i m a l C a r e ( C C A C ) , the b o d y that governs a n i m a l research i n C a n a d a , stipulates i n their E t h i c s o f A n i m a l Investigation g u i d e l i n e s ( 1 9 8 9 , p . l ) that research a n i m a l s " m u s t not be subjected to unnecessary p a i n or d i s t r e s s " a n d " i f p a i n or distress is necessary c o n c o m i t a n t to the study, it m u s t be m i n i m i z e d b o t h i n intensity and d u r a t i o n " . T h e r e f o r e , w h e r e m u l t i p l e methods are a v a i l a b l e for a c h i e v i n g the same a i m s , the m e t h o d that causes the least p a i n and distress s h o u l d be used. F u r t h e r m o r e , w h e r e a p a i n f u l or distressful procedure is required to meet the a i m s o f the study, efforts m u s t be m a d e to mitigate these effects. A l t h o u g h these g u i d e l i n e s a l l o w for p r a g m a t i c concerns to be addressed, they require that a n i m a l p a i n and distress be m i n i m i z e d to the greatest extent p o s s i b l e w i t h i n these c o n f i n e s . In order to meet this requirement, w e m u s t first determine w h i c h methods cause p a i n and distress, a n d then determine h o w these states c a n be m i n i m i z e d . T o assess whether k i l l i n g methods cause p a i n and distress w e c a n e x a m i n e three lines o f evidence:  1) h u m a n  self-report  data to  determine  what  subjective  states are  potentially  associated w i t h e x p o s u r e , 2) p h y s i o l o g i c a l responses to observe the m e t h o d ' s potential for a c t i v a t i n g n o c i c e p t o r s or n e u r o - e n d o c r i n e stress responses, and 3) b e h a v i o u r a l data to assess whether the m e t h o d results i n behaviours i n d i c a t i v e o f p a i n or distress, and to determine the l e v e l o f a v e r s i o n associated w i t h the s t i m u l u s . A l t h o u g h it m a y not be p o s s i b l e to c o m p l e t e l y prevent p a i n and distress, it s h o u l d be p o s s i b l e to i d e n t i f y those procedures that result i n the least p a i n a n d distress, a n d thus p r o v i d e a basis f o r r e c o m m e n d a t i o n s that c a n be used b y a n i m a l caregivers.  2  1.2 Carbon Dioxide Euthanasia C a r b o n d i o x i d e (CO2) is the m o s t w i d e l y u s e d euthanasia agent for laboratory rodents. In CO2 euthanasia o f rodents, the a n i m a l is either p l a c e d into a c h a m b e r that is p r e - f i l l e d w i t h CO2 at a c o n c e n t r a t i o n o f greater than 7 0 % , or the a n i m a l is p l a c e d i n a n empty c h a m b e r that is then g r a d u a l l y f i l l e d w i t h CO2. A l t h o u g h s o m e p r e v i o u s w o r k has e x a m i n e d whether CO2 euthanasia causes distress i n rats, the results to date have b e e n i n c o n c l u s i v e .  1.2.1 M o d e o f a c t i o n Although  CO2  is categorized as a non-anaesthetic gas, it does have  anaesthetic  properties. In the rat, it causes a decrease i n b r a i n e x c i t a b i l i t y at concentrations as l o w as 5 % , light anaesthesia b e g i n n i n g at 2 5 % , and deeper anaesthesia at a p p r o x i m a t e l y 4 0 % ( r e v i e w e d b y W o o d b u r y et a l . , 1958). H u m a n s s h o w e l e c t r o e n c e p h a l o g r a m ( E E G ) depression and are unable to carry out s i m p l e c o m m a n d s w h e n breathing 20%  C0  2  ( M e y e r et a l . , 1966). D e a t h c a n o c c u r  at concentrations o f a p p r o x i m a t e l y 3 0 % CO2 and higher ( D a n n e m a n et a l . , 1 9 9 7 ; Sharp et a l . , 2 0 0 6 ) , l i k e l y due to depression o f b r a i n centers responsible for c i r c u l a t i o n and r e s p i r a t i o n ; the exact CO2 c o n c e n t r a t i o n necessary f o r death depends o n the d u r a t i o n o f exposure ( D a n n e m a n et a l , 1997). D u r i n g m e t a b o l i s m , the b o d y uses o x y g e n (O2) a n d p r o d u c e s CO2 and these t w o gases are constantly e x c h a n g e d b y the respiratory s y s t e m . A i r contains a p p r o x i m a t e l y 0 . 0 3 % CO2 and 2 0 . 9 % O2 (and 7 8 % nitrogen), but due to m e t a b o l i s m , the tissues c o n t a i n higher l e v e l s o f CO2 and l o w e r l e v e l s o f O2. T h i s results i n a gas concentration gradient b e t w e e n the air and the b l o o d at the lungs that a l l o w s f o r the uptake o f O2 into the b l o o d and release o f CO2 into the air, and b e t w e e n the b l o o d and the tissues i n the rest o f the b o d y that a l l o w s for the release o f O2 to the tissues a n d the uptake o f C 0  2  into the b l o o d . B e c a u s e o f this gradient, C 0  3  2  generally occurs  only at low concentrations (<6%) in the body. Total CO2 content in the body consists o f carbamino compounds, CO2, bicarbonate ions (HCO3") and carbonic acid (H2CO3). The last three exist in the following equilibrium:  C 0 + H 0 «-+ H2CO3 2  2  H + HCO3" +  A s the concentration o f CO2 in the air is increased, it builds up in the tissues and shifts this equilibrium to the right, producing hydrogen ions and reducing p H . Although the body has adaptations that increase the removal o f C 0  2  at the lungs under normal conditions, the  concentrations o f CO2 used in euthanasia are far beyond the body's compensatory mechanisms. Furthermore, the blood brain barrier is very permeable to CO2, and the C S F has little buffering capacity. This results in a greater drop in p H in the C S F than in the blood and other tissues (Brodie & Woodbury, 1958), and it is thought that this reduction i n p H causes CO2 narcosis and death (e.g. Brodie & Woodbury 1958; Eisele et al., 1967; Martoft et al. 2003; Meyer et al., 1961; Woodbury et al., 1958). Specifically, these changes in C S F p H are thought to reduce neuron excitability. For example, in vitro studies have demonstrated that a reduction i n extracellular p H due to exposure 20% CO2 reduces the activity o f neurons in the hippocampus via a p H dependent mechanism (e.g. Dulla et al., 2005; H s u et al., 2000; Lee et al., 1996; Velisek, 1998). The exact mechanisms for these effects on neuron functioning are not currently known, but p H effects are thought to be mediated by alterations in cell ion gradients (e.g. Gifford et al., 1990; Pasternack et al., 1992; Tang et al., 1990; Tombaugh and Somjen, 1996), possibly through reconfiguration o f relevant proteins associated with cell ion channels, receptors or enzymes (Somero, 1986).  4  1.2.2 Time-course of CO2 euthanasia in rats A n u m b e r o f studies have observed the t i m e course o f CO2 euthanasia i n rats, and this i n f o r m a t i o n p r o v i d e s a n i n d i c a t i o n o f the d u r a t i o n o f exposure and the m a x i m u m concentrations o f CO2 that o c c u r w h i l e the a n i m a l is s t i l l c o n s c i o u s . These results have been obtained for pre1  f i l l a n d g r a d u a l - f i l l CO2  euthanasia o f rats. A u t h o r s  differ  i n their  criteria for  loss  of  c o n s c i o u s n e s s , but the m a j o r i t y o f experiments use onset o f a t a x i a to indicate the i n i t i a l depression o f the central nervous system ( C N S ) , a n d c o m p l e t e loss o f posture to indicate loss o f c o n s c i o u s n e s s . S o m e studies e x a m i n i n g species other than rats have u s e d E E G s  (Raj  and  G r e g o r y , 1 9 9 4 ; R a j et a l . , 1 9 9 7 ; R a j et a l . , 1 9 9 8 ; M a r t o f t et a l . , 2 0 0 2 ) , and somatosensorye v o k e d potentials ( S E P s ) (Raj and G r e g o r y , 1 9 9 4 ; R a j et a l . , 1 9 9 7 ; M a r t o f t et a l . , 2 0 0 2 ) to g a i n a m o r e accurate estimate o f tinie to u n c o n s c i o u s n e s s , and there appears to be a slight t i m e l a g between loss o f posture and changes i n E E G s and S E P s ( C o e n e n et a l . , 2 0 0 0 ; R a j et a l . , 1992). H e w e t t et a l . (1993) f o u n d that d u r i n g g r a d u a l - f i l l CO2 exposure at a f l o w rate o f 2 0 % per m i n u t e , rats s h o w e d i m m o b i l i t y after 72 s and loss o f r i g h t i n g r e f l e x after 109 s.  However,  i m m o b i l i t y w a s o n l y d e f i n e d as i n a c t i v i t y so the delay between c o m p l e t e loss o f posture and l o s s o f r i g h t i n g r e f l e x w o u l d have been shorter. In fact, C o e n e n et a l . ( 1 9 9 5 ) f o u n d that c o m p l e t e loss o f posture i n rats w a s w e l l correlated w i t h onset o f a f u l l y aberrant E E G , and that the delay w a s less than 10 s d u r i n g g r a d u a l - f i l l CO2 exposure. F o r the sake o f s i m p l i c i t y , ataxia and loss o f posture w i l l be used to s u m m a r i z e the duration data for each o f the different CO2 m e t h o d s , w i t h the a s s u m p t i o n that these measures represent the onset o f loss o f consciousness and that c o m p l e t e i n s e n s i b i l i t y l i k e l y f o l l o w s shortly afterwards. .  The term 'conscious' is used here according to what Block (1995) has referred to as "phenomenal consciousness". This refers to awareness and the ability to experience events, but does not imply being able to report on this experience or of being self-conscious. 'Conscious' is used throughout this dissertation to indicate when the animal is awake and therefore is presumably capable of experiencing negative affective states. 1  5  D u r i n g p r e - f i l l euthanasia i n rats w i t h CO2 concentrations greater than 7 0 % , t i m e to loss o f posture generally ranges from 7 s to 2 0 s ( B l a c k s h a w et a l . , 1 9 8 8 ; K o h l e r et a l . , 1 9 9 9 ; H e w e t t et a l . , 1 9 9 3 ; D a n n e m a n et a l . , 1 9 9 7 ; C o e n e n et a l . , 1 9 9 5 ; B r i t t , 1987), a n d a t a x i a is not generally o b s e r v e d because o f the speed o f c o l l a p s e . W i t h a fast f l o w rate (>50%» o f the c h a m b e r v o l u m e b e i n g added per m i n u t e ) , a t a x i a occurs at 13 s to 18 s and loss o f posture at 2 6 s to 4 8 s ( C o e n e n et a l . , 1 9 9 5 ; S m i t h and H a r r a p , 1997). W i t h a m e d i u m f l o w rate ( 1 5 % to 5 0 % o f the c h a m b e r v o l u m e added per m i n u t e ) a t a x i a ranges f r o m 4 2 s to 120 s, and loss o f posture ranges f r o m 9 0 s to 120 s (Hornett and H a y n e s , 1 9 8 4 ; H a c k b a r t h et a l . , 1 9 9 9 ; S m i t h and H a r r a p , 1 9 9 7 ; D a n n e m a n et a l . , 1997). S l o w f i l l rates ( < 1 5 % o f the c h a m b e r v o l u m e added per m i n u t e ) take m u c h longer, w i t h a t a x i a o c c u r r i n g at 120 s to 180 s and loss o f posture o c c u r r i n g at 120 s to 2 4 0 s (Hornett a n d H a y n e s , 1984). L i t t l e is k n o w n about the concentrations o f CO2 that are r e q u i r e d to cause loss  of  c o n s c i o u s n e s s d u r i n g g r a d u a l - f i l l CO2 euthanasia. W i t h a m e d i u m CO2 f l o w rate, rats have been f o u n d to lose consciousness at concentrations as l o w as 4 0 % ( S m i t h and H a r r a p , 1997). T h e majority  of  studies  examining  gradual-fill  CO2  exposure  have  not  monitored  concentrations, so it is not clear whether f l o w rate has a n effect o n the CO2  CO2  concentration  needed to cause loss o f consciousness i n rats. H o w e v e r , results i n m i c e suggest that a m e d i u m flow  rate o f 3 0 % per m i n u t e is associated w i t h a l o w e r concentration o f CO2 at loss  of  c o n s c i o u s n e s s than a fast flow rate o f 6 0 % per m i n u t e ( A m b r o s e et a l . , 2 0 0 0 ) . W h i l e the cause o f this effect is u n k n o w n , the longer d u r a t i o n o f CO2 exposure w i t h s l o w f i l l rates m a y a l l o w m o r e t i m e for p H adjustments to o c c u r i n the b l o o d and C S F . In s u m m a r y , p r e - f i l l CO2 exposure takes less t i m e to cause loss o f consciousness than g r a d u a l - f i l l e x p o s u r e , but a n i m a l s are e x p o s e d to higher C 0  2  concentrations d u r i n g the p e r i o d o f  c o n s c i o u s n e s s . W i t h g r a d u a l - f i l l CO2 exposure, faster flow rates cause a shorter d u r a t i o n to loss  6  o f c o n s c i o u s n e s s , but m a y b e associated w i t h higher CO2 concentrations at the t i m e o f loss o f consciousness.  1.2.3 Contexts of CO2 use C a r b o n d i o x i d e is r e c o m m e n d e d f o r euthanasia o f laboratory rodents i n m a n y western countries s u c h as C a n a d a ( C C A C , 1993) the U S A ( A V M A , 2 0 0 0 ) , the U K ( U K H o m e O f f i c e , 1997), the countries o f the E u r o p e a n U n i o n ( C l o s e et a l . , 1997) a n d A u s t r a l i a and N e w Z e a l a n d ( A N Z C C A R T , 1993). T h e r e c o m m e n d e d m e t h o d o f d e l i v e r y v a r i e s , but i n general, gradual i n d u c t i o n is r e c o m m e n d e d over p r e - f i l l . A l t h o u g h some r e c o m m e n d a t i o n s stipulate a f l o w rate for g r a d u a l i n d u c t i o n , anecdotal reports suggest that f e w f a c i l i t i e s m o n i t o r f l o w rate. C a r b o n d i o x i d e is also used f o r euthanasia o f i n j u r e d w i l d l i f e i n r e h a b i l i t a t i o n centres and f o r st un ning  or k i l l i n g  o f farmed mink, pigs,  poultry,  a n d f i s h ( v i a water  bath).  R e c o m m e n d a t i o n s f o r use o f CO2 w i t h these species d i f f e r f r o m those a p p l i c a b l e to laboratory a n i m a l s i n that they often require p r e - f i l l i n g or r a p i d l y f i l l i n g the c h a m b e r rather than gradual f i l l . F o r e x a m p l e , the U K W e l f a r e o f A n i m a l s (Slaughter  or K i l l i n g )  Regulation  (1995)  stipulates that f o r the st unn in g o f p i g s , the c h a m b e r must reach a c o n c e n t r a t i o n o f at least 7 0 % i n less than 3 0 seconds, a n d f o r the k i l l i n g o f m i n k , the a n i m a l m u s t be p l a c e d into a c h a m b e r c o n t a i n i n g 1 0 0 % C 0 ( U K H o m e O f f i c e , 1995). I n contrast, the U K C o d e o f P r a c t i c e f o r k i l l i n g 2  o f e x p e r i m e n t a l a n i m a l s r e c o m m e n d s exposure to a gradual increase i n CO2 ( U K H o m e O f f i c e , 1997). H o w e v e r , there has been n o research to j u s t i f y the use o f different d e l i v e r y methods f o r different species.  1.2.4 Advantages of CO2 euthanasia T h e r e are a n u m b e r o f reasons f o r the w i d e s p r e a d use o f CO2 euthanasia o f laboratory a n i m a l s . A n e c d o t a l reports suggest that there i s a general p e r c e p t i o n w i t h i n the s c i e n t i f i c 7  c o m m u n i t y that the effects o f CO2 o n e x p e r i m e n t a l results are k n o w n , and that u n k n o w n effects o n e x p e r i m e n t a l results m a y be i n t r o d u c e d b y c h a n g i n g to other procedures. Initial experiments o n the use o f CO2 for k i l l i n g o c c u r r e d i n the late 1 8 0 0 ' s ( r e v i e w e d b y H i l l and F l a c k , 1908), and there is therefore a l o n g history o f CO2 use. T h e r e have also been a n u m b e r o f studies c o n d u c t e d to determine the effects o f CO2 o n s p e c i f i c metabolites and tissues, i n order to ensure that experimental  results  are  not  affected  (e.g.  Fawell  et  al.,  1 9 7 2 ; Pecaut  et  al.,  2000;  B e r g e r s w e e n e y et a l . , 1994). In situations where a c o n f o u n d i n g effect o n tissue measures is e x p e c t e d , s u c h as d u r i n g t o x i c o l o g i c a l research, other euthanasia methods l i k e decapitation and c e r v i c a l d i s l o c a t i o n are often e m p l o y e d . CO2 also has a n u m b e r o f advantages f o r the operator i n c o m p a r i s o n to other methods. B e c a u s e it c a n be d e l i v e r e d i n a c h a m b e r , it i n v o l v e s little h a n d l i n g o f a n i m a l s and c a n be used to q u i c k l y euthanize large n u m b e r s o f a n i m a l s at a t i m e . In c o m p a r i s o n to p h y s i c a l methods and injectable anaesthetics, it requires c o n s i d e r a b l y less t i m e . T h i s m e t h o d also requires little direct interaction w i t h a n i m a l s and results i n f e w effects that m a y be d i s t u r b i n g to the operator. A s a respiratory gas, CO2 is also safer for the operator than other gas euthanasia agents such as c a r b o n m o n o x i d e , c h l o r o f o r m , ether and gas anaesthetics, w h i c h c a n pose si g n i f i cant health risks. CO2 also has cost advantages i n c o m p a r i s o n to other m e t h o d s . D e c a p i t a t i o n and c e r v i c a l d i s l o c a t i o n require m i n i m a l e q u i p m e n t , but have considerable costs i n terms o f e m p l o y e e t i m e . O t h e r p h y s i c a l methods s u c h as m i c r o w a v e i r r a d i a t i o n require the purchase o f  expensive  e q u i p m e n t . A n a e s t h e t i c s tend to be m u c h m o r e expensive than CO2. T h e r e are also indirect costs f o r anaesthetic m e t h o d s , s u c h as the purchase o f needles (for injectable anaesthetics), and anaesthetic m a c h i n e s and s c a v e n g i n g systems (for gaseous anaesthetics). W i t h respect to the w e l f a r e o f a n i m a l s CO2 p r o v i d e s m a n y benefits i n c o m p a r i s o n to other euthanasia m e t h o d s . M o s t i m p o r t a n t l y , CO2 i s easy to d e l i v e r p r o p e r l y w i t h little training. 8  P h y s i c a l m e t h o d s , injectable anaesthetics and inert gases have the potential to be a d m i n i s t e r e d i n c o r r e c t l y and to result i n considerable p a i n and distress for the a n i m a l . G a s e s i n general are also advantageous because they require m i n i m a l h a n d l i n g o f the a n i m a l . L a b o r a t o r y rodents are not generally habituated to regular h a n d l i n g and restraint, a n d these procedures c a n therefore cause c o n s i d e r a b l e distress (e.g. Sharp et a l . , 2 0 0 2 , 2 0 0 3 ) . F u r t h e r m o r e , injections into the peritoneal c a v i t y  c a n be p a i n f u l , and injectable anaesthetics c a n be  irritating,  possibly  s t i m u l a t i n g v i s c e r a l nociceptors. A l t h o u g h CO2 has m a n y advantages o v e r other euthanasia m e t h o d s , there has been o n g o i n g debate as to whether it causes p a i n and distress i n laboratory rodents.  1.3 Animal Distress and Carbon Dioxide 1.3.1 What is animal distress? A n i m a l w e l f a r e is generally c o n s i d e r e d to be enhanced b y g o o d health, p o s i t i v e affect, and the a b i l i t y to p e r f o r m natural b e h a v i o u r s , and reduced b y p o o r health, negative affect and inadequate outlets for b e h a v i o u r (Fraser et a l . , 1997). W i t h a procedure s u c h as CO2 euthanasia, the m a i n c o n c e r n is w i t h the affective state o f the a n i m a l . In d e s c r i b i n g e v e r y d a y usage, T h e C a n a d i a n O x f o r d D i c t i o n a r y (2000) defines distress as "severe trouble, anxiety, s o r r o w " (p.275), i n d i c a t i n g a negative e m o t i o n a l state. S e l y e (1975) w a s the first to define distress s c i e n t i f i c a l l y , and he used this t e r m to refer to b i o l o g i c a l responses to negative stressors, where  stressor refers to a n actual or p e r c e i v e d threat to  h o m e o s t a s i s . W i t h s p e c i f i c reference to a n i m a l w e l f a r e , the t e r m distress has b e e n d e f i n e d i n a n u m b e r o f different w a y s ; h o w e v e r , i n general there is agreement that 'distress' refers to a negative state that d e v e l o p s w h e n an o r g a n i s m is unable to adapt to a stressor (e.g. K i t c h e n et a l . , 1 9 8 7 ; M o b e r g , 2 0 0 0 ; N R C , 2 0 0 3 ; R o w a n et a l . , 1998). T h i s d e f i n i t i o n , therefore, acts as a n  9  u m b r e l l a term that encompasses negative affect associated w i t h m o r e s p e c i f i c negative states s u c h as p a i n , d i s c o m f o r t and fear.  1.3.2 Potential mechanisms for CO2 distress T h e r e are three m a i n m e c h a n i s m s b y w h i c h CO2 m i g h t cause distress i n rats. T h e rationale f o r the first t w o m e c h a n i s m s that are d i s c u s s e d i s l a r g e l y dependent o n h u m a n experiences d u r i n g CO2 exposure. I f h u m a n s report p a i n o r distress d u r i n g CO2 exposure, then f o l l o w i n g the l o g i c o f D a w k i n s (1980) it c a n be h y p o t h e s i z e d that CO2 has the potential f o r s i m i l a r sensations i n a n i m a l s w i t h s i m i l a r a n a t o m y a n d p h y s i o l o g y . h i g h e r order c o g n i t i v e  A l t h o u g h h u m a n s have  p r o c e s s i n g that c a n affect the q u a l i t y o f a sensation, the general  p e r c e p t i o n o f a s t i m u l u s as appetitive or aversive is l i k e l y c o n s e r v e d b e t w e e n h u m a n s and other m a m m a l s . H o w e v e r , it i s important to r e c o g n i z e that sensations m a y also d i f f e r  between  different species, since e a c h i s adapted to a particular n i c h e .  1.3.2.1 Pain induced by carbonic acidformation T h e m o s t w i d e l y d i s c u s s e d m e c h a n i s m f o r distress d u r i n g CO2 euthanasia i s that CO2 c a n f o r m c a r b o n i c a c i d o n nasal m u c o u s m e m b r a n e s , a n d this stimulates t r i g e m i n a l nociceptors and causes p a i n (e.g. L e a c h et a l . , 2002a). T h i s c a n be extended b y c o n s i d e r i n g that CO2 also has the potential to affect the c o r n e a and c o n j u n c t i v a o f the eyes, and p o s s i b l y chemoreceptors i n the l o w e r respiratory tract that are sensitive to other irritants, al th oug h this has n o t been f u l l y examined. A n u m b e r o f studies h a v e assessed the potential f o r CO2 to activate nociceptors a n d e v o k e p a i n i n h u m a n s w h e n a p p l i e d to the nasal m u c o s a , c o r n e a and c o n j u n c t i v a . N e g a t i v e nasal m u c o s a l potentials have been r e c o r d e d f r o m the nasal s e p t u m a n d u s e d as a n o n - i n v a s i v e m e t h o d f o r measurement o f t r i g e m i n a l n o c i c e p t o r a c t i v a t i o n . N e g a t i v e m u c o s a l potentials " a r e 10  thought to be the result o f s u m m a t i n g receptor potentials o f c h e m o s e n s i t i v e n o c i c e p t o r s o f the t r i g e m i n a l n e r v e " ( T h u r a u f et a l . , 1 9 9 3 , p.293). T h e s e potentials have b e e n r e c o r d e d i n response to CO2 concentrations o v e r 4 5 % , and have been f o u n d to increase w i t h CO2 concentration and a p p l i c a t i o n duration (Thurauf, 1993). H u m a n self-report data indicate that CO2 is detectable at the nasal m u c o s a at concentrations o f o n l y 2 0 % , and that it b e c o m e s o v e r t l y p a i n f u l at concentrations above a p p r o x i m a t e l y 5 0 % ( A n t o n et a l . , 1 9 9 2 ; T h u r a u f et a l . , 2 0 0 2 ) . H o w e v e r , the c o n c e n t r a t i o n that is p e r c e i v e d as p a i n f u l does v a r y , w i t h the p a i n t h r e s h o l d r a n g i n g f r o m 3 2 . 5 % to 5 5 % d e p e n d i n g o n the i n d i v i d u a l ( A n t o n et a l . , 1992). D a n n e m a n et a l . (1997) h a d subjects rate the n o x i o u s n e s s o f CO2 at concentrations r a n g i n g f r o m 5 0 % to 1 0 0 % w h e n i n h a l e d v i a the nose, and f o u n d that the m a j o r i t y o f subjects rated 50%) CO2 as unpleasant  or  u n c o m f o r t a b l e , a n d 1 0 0 % CO2 as p a i n f u l . F u r t h e r m o r e , at each c o n c e n t r a t i o n some subjects felt they c o u l d not take a c o m p l e t e breath, and the w o r d s t i n g l i n g , p r i c k l i n g a n d b u r n i n g were used to describe the sensation at a l l concentrations. In rats, CO2 has b e e n s h o w n to activate dorsal h o r n neurons that r e c e i v e input f r o m t r i g e m i n a l n o c i c e p t o r s i n the nasal m u c o s a ( A n t o n et a l . , 1 9 9 1 ; P e p p e l and A n t o n ,  1993),  suggesting that CO2 also has the potential to cause p a i n i n rats. P e p p e l a n d A n t o n (1993) f o u n d that rat n o c i c e p t o r s r e s p o n d to CO2 concentrations that are s i m i l a r to those p r e v i o u s l y reported f o r h u m a n n o c i c e p t o r s . CO2 concentrations b e l o w 3 7 % were subthreshold f o r the m a j o r i t y o f rat neurons, a n d the C 0  2  n o c i c e p t i o n t h r e s h o l d ranged f r o m 3 7 % to 5 0 % CO2 for the m a j o r i t y o f  neurons. F u r t h e r m o r e , the graded response tended to increase i n a linear f a s h i o n u n t i l saturation w a s reached at a p p r o x i m a t e l y 8 7 % CO2. A n o t h e r m e t h o d o f assessing the potential for a substance to e l i c i t p a i n at the upper respiratory tract is t h r o u g h its a b i l i t y to e l i c i t changes i n cardiorespiratory r h y t h m s .  Inhaled  irritants are k n o w n to i n d u c e a r e f l e x apnea and heart rate r e d u c t i o n , and these responses are thought to reduce transfer o f h a r m f u l substances into the b o d y ( W i d d i c o m b e , 1986). In rats, 11  1 0 0 % CO2 e l i c i t s this apnea and b r a d y c a r d i a , but CO2 concentrations o f 10, 2 5 and 5 0 % do not ( Y a v a r i et a l . , 1996). T h e t h r e s h o l d necessary for e l i c i t i n g cardiorespiratory alterations w a s not d e t e r m i n e d , but this data suggests that C 0  2  concentrations less than 50% are not irritating to the  nasal m u c o s a i n rats. T h e effect o f CO2 o n p a i n and a c t i v a t i o n o f n o c i c e p t o r s i n the c o r n e a and c o n j u n c t i v a appears to be s i m i l a r to its effect at the nasal m u c o s a . F e n g and S i m p s o n (2003) e x a m i n e d p a i n thresholds i n h u m a n s i n response to CO2 a p p l i c a t i o n to the c o r n e a a n d c o n j u n c t i v a and f o u n d that they o c c u r r e d at 31 % and 5 4 % , respectively. P a r t i c i p a n t s a g a i n characterized the sensation at t h r e s h o l d as b u r n i n g or s t i n g i n g . In another study e x a m i n i n g the effect o f CO2 o n the c o r n e a , subjects reported m i l d s t i n g i n g at a m e a n CO2 concentration o f 33.5%, and overt p a i n at  47.5%  ( C h e n et a l . , 1995). C h e n et a l . also e x a m i n e d the responses o f cat c o r n e a l nociceptors to CO2 a p p l i c a t i o n a n d f o u n d that they r e s p o n d e d at a m e a n t h r e s h o l d o f 4 0 % C 0 , w h i c h is s i m i l a r to 2  the p a i n responses o b s e r v e d i n h u m a n s . A c t i v a t i o n o f rat c o r n e a l n o c i c e p t o r s b y CO2 has also b e e n demonstrated ( H i r a t a et a l . , 1999), a l t h o u g h the t h r e s h o l d l e v e l o f C 0  2  needed to e l i c i t  n o c i c e p t o r a c t i v a t i o n w a s not i d e n t i f i e d . T h i s e v i d e n c e suggests that CO2 has the potential to cause p a i n i n rats  through  s t i m u l a t i o n o f n o c i c e p t o r s i n the nasal m u c o s a and cornea. P a i n is l i k e l y to o c c u r w i t h p r e - f i l l m e t h o d s i n w h i c h a c o n s c i o u s a n i m a l is e x p o s e d to h i g h concentrations o f CO2 ( > 7 0 % ) . D u r i n g g r a d u a l f i l l CO2 euthanasia a n i m a l s reach u n c o n s c i o u s n e s s at about 40%  ( S m i t h and H a r r a p ,  1997), so there is some potential for l o w l e v e l s o f p a i n to o c c u r a r o u n d the t i m e o f loss o f consciousness.  1.3.2.2 Non-pain discomfort associated with hypercapnia and hypoxia Distress d u r i n g CO2 euthanasia c o u l d also o c c u r t h r o u g h the effects o f h y p e r c a p n i a (elevated b l o o d CO2) and h y p o x i a (reduced b l o o d O2), w h i c h have b e e n s h o w n to cause d y s p n e a 12  in  humans. Dyspnea  is  a n unpleasant  sensation o f  breathlessness  which  is s o m e t i m e s  a c c o m p a n i e d b y other negative sensations s u c h as headache, f l u s h , restlessness, heart p o u n d i n g , d r o w s i n e s s , a n d d i z z i n e s s (e.g. M o o s a v i et a l . , 2 0 0 3 ) . W i t h m i l d increases i n i n s p i r e d CO2 and decreases i n i n s p i r e d O2, increased v e n t i l a t i o n results i n a r e d u c t i o n or e l i m i n a t i o n o f d y s p n e a (Banzett et a l . , 1 9 9 6 ; F o w l e r , 1 9 5 7 ; S h e a et a l . , 1996), but there are l i m i t s to this c o m p e n s a t o r y m e c h a n i s m s u c h that  dyspnea  s t i l l occurs  during  spontaneous  breathing w i t h  moderate  h y p e r c a p n i a and h y p o x i a (Shea et a l . , 1996). N u m e r o u s studies have demonstrated that h y p e r c a p n i a results i n d y s p n e a i n h u m a n s d u r i n g spontaneous breathing (e.g. B a n z e t t et a l . , 1 9 9 6 ; S h e a et a l . , 1996). M o s t studies e x a m i n i n g d y s p n e a due to h y p e r c a p n i a have m o n i t o r e d e n d - t i d a l p a r t i a l pressures (PET) o f CO2 (concentration o f CO2 breathed out) rather than i n s p i r e d l e v e l s o f CO2, and it is therefore d i f f i c u l t to determine the i n s p i r e d concentrations o f CO2 that result i n different l e v e l s  of  d y s p n e a . H o w e v e r , s o m e studies have e x a m i n e d i n s p i r e d CO2 l e v e l s a n d c a n p r o v i d e insight into the CO2 concentrations necessary to e l i c i t d y s p n e a . L i o t t i et a l . ( 2 0 0 1 ) h a d subjects rate their sensations o f d y s p n e a o n a 100 p o i n t scale d u r i n g exposure to 8 % CO2 i n O2, a n d d y s p n e a l e v e l s were rated as either 73 or 55 d e p e n d i n g o n whether the gas m i x t u r e w a s d e l i v e r e d u s i n g a f a c e m a s k or a m o u t h p i e c e , respectively. D r i p p s and C o m r o e ( 1 9 4 7 ) f o u n d that a p p r o x i m a t e l y 3 0 % o f study participants reported d y s p n e a w h e n e x p o s e d to either 7.6 or 1 0 . 4 % CO2 i n O2. In the late 1 8 0 0 ' s and early 1 9 0 0 ' s researchers often u s e d themselves as e x p e r i m e n t a l subjects i n the i n v e s t i g a t i o n o f respiratory p h y s i o l o g y ( s u m m a r i z e d b y H i l l and F l a c k , 1908). T h e s e studies p r o v i d e insight into the concentrations o f CO2 that result i n moderate to severe d y s p n e a d u r i n g spontaneous breathing. A c c o r d i n g to H i l l and F l a c k ( 1 9 0 8 ) , G r e e n w o o d f o u n d that he w a s able to breathe a m i x t u r e o f 15.3%  CO2 and 1 4 . 5 % O2 w i t h m a r k e d d y s p n e a , but  that h i g h e r l e v e l s o f CO2 resulted i n closure o f the glottis a n d prevented i n h a l a t i o n . H a l d a n e and S m i t h ( 1 8 9 2 ) f o u n d that w h e n breathing 1 8 . 6 % CO2 i n air, they d e v e l o p e d p r o f o u n d d y s p n e a i n 13  one to t w o m i n u t e s that w a s a c c o m p a n i e d b y t h r o b b i n g i n the head a n d m e n t a l d u l l n e s s . T o g e t h e r w i t h the results f r o m m o d e r n studies, these results indicate that d u r i n g spontaneous b r e a t h i n g , d y s p n e a a n d other negative sensations c a n o c c u r w i t h C O 2 concentrations as l o w as 8%, a n d that severe d y s p n e a o c c u r s at C O 2 concentrations greater than a p p r o x i m a t e l y 1 5 % . H y p o x i a also appears to have the potential to cause d y s p n e a i n h u m a n s , but to a lesser extent than h y p e r c a p n i a . M o o s a v i et a l . ( 2 0 0 3 ) f o u n d that d u r i n g  constrained breathing,  participants reported onset o f d y s p n e a w h e n PETO2 decreased b e l o w a p p r o x i m a t e l y 6 0 T o r r (PETO2 l e v e l s w e r e 108 T o r r d u r i n g baseline w h e n breathing air). H o w e v e r , at the study's ethical PETO2 l i m i t o f 4 0 T o r r (the l o w e s t i n s p i r e d O2 tested w a s 7%) the m a j o r i t y o f participants rated their d y s p n e a as less than 4 0 o n a v i s u a l analogue scale w h i c h ranged f r o m 0 (no dyspnea) to 100 (extreme dyspnea). T h i s indicates that O2 concentrations o f less t h a n 7 % are needed to e v o k e moderate sensations o f d y s p n e a i n h u m a n s d u r i n g c o n s t r a i n e d b r e a t h i n g . T h e potential f o r dyspnea  due to h y p o x i a  alone  i n spontaneously  breathing  humans  has not been  fully  investigated. H y p o x i a - i n d u c e d loss o f c o n s c i o u s n e s s o c c u r s o c c a s i o n a l l y i n p i l o t s as a result o f c a b i n p r e s s u r i z a t i o n f a i l u r e ( C a b l e , 2 0 0 3 ) , suggesting that s p o n t a n e o u s l y b r e a t h i n g h u m a n s d o not e x p e r i e n c e d y s p n e a p r i o r to loss o f consciousness w i t h h y p o x i a . H o w e v e r , h y p o x i a has a synergistic effect o n ventilatory responses to h y p e r c a p n i a (e.g. N i e l s o n a n d S m i t h , 1952), a n d augments sensations o f d y s p n e a due to h y p e r c a p n i a i n h u m a n s ( B a n z e t t et a l . , 1 9 9 6 ; M a s u d a et a l , 2 0 0 1 ) . T h e s e results indicate that h y p o x i a alone is u n l i k e l y to cause sensations o f d y s p n e a d u r i n g free b r e a t h i n g , but that it m a y increase d y s p n e a due to h y p e r c a p n i a . T h u s , i f d y s p n e a o c c u r s d u r i n g C 0 euthanasia, h y p o x i a m a y be a c o n t r i b u t i n g factor. 2  T o date, n o m o d e l s h a v e been d e v e l o p e d f o r the assessment o f d y s p n e a i n c o n s c i o u s a n i m a l s , so it is not k n o w n whether h y p e r c a p n i a a n d h y p o x i a cause d y s p n e a i n rats. D y s p n e a i n rats m a y be i n d i c a t e d b y increases i n b r e a t h i n g depth a n d f r e q u e n c y . T h e t e r m d y s p n e a is s o m e t i m e s u s e d i n the veterinary literature to refer d i r e c t l y to these b r e a t h i n g changes (e.g. 14  H o r n e t t and H a y n e s , 1984), but ' d y s p n e a ' actually refers to a sensation o f breathlessness rather t h a n the p h y s i c a l changes i n breathing that s o m e t i m e s a c c o m p a n y this sensation. W h i l e increases i n breathing ( s o m e t i m e s referred to as g a s p i n g or l a b o u r e d breathing) have b e e n o b s e r v e d i n rats d u r i n g CO2 euthanasia, it is not clear whether they o c c u r before or after loss o f c o n s c i o u s n e s s , and whether they are m o r e severe w i t h p r e - f i l l or g r a d u a l - f i l l exposure ( C o e n e n et a l . , 1 9 9 5 ; H o r n e t t and H a y n e s , 1 9 8 4 ; I w a r s s o n & R e h b i n d e r , 1 9 9 3 ; S m i t h a n d H a r r a p , 1997). F u r t h e r m o r e , h u m a n m e d i c a l studies suggest that breathing alterations and sensations  of  d y s p n e a do not a l w a y s o c c u r concurrently ( L u s h et a l . , 1988), w h i c h brings into question the usefulness o f this measure for assessing d y s p n e a i n rats. W h i l e there is n o e v i d e n c e c o n f i r m i n g that d y s p n e a o c c u r s i n rats, it is reasonable to assume that s u c h a b a s i c response to changes i n air c o m p o s i t i o n and b l o o d gas l e v e l s w o u l d be c o n s e r v e d b e t w e e n species. If so, b o t h p r e - f i l l (<6%  O2, > 7 0 % CO2) a n d g r a d u a l - f i l l CO2  euthanasia ( < 1 2 % 0 , > 4 0 % CO2) have the potential to cause strong sensations o f d y s p n e a i n 2  rats, m a i n l y due to h y p e r c a p n i a .  1.3.2.3 Fear due to novelty N o v e l t y has b e e n suggested to i n d u c e an a p p r o a c h - a v o i d a n c e c o n f l i c t i n rats, resulting f r o m a n interaction b e t w e e n exploratory m o t i v a t i o n and fear ( M o n t g o m e r y , 1955). CO2 c o u l d therefore cause distress i n rats b y a c t i n g as a n o v e l s t i m u l u s that e l i c i t s fear. O d o u r p e r c e p t i o n occurs as a result o f b o t h o l f a c t o r y a n d t r i g e m i n a l s t i m u l a t i o n (e.g. C a i n a n d M u r p h y , 1980). W h i l e CO2 is thought to stimulate m a i n l y t r i g e m i n a l neurons, h u m a n s p e r c e i v e a n odour q u a l i t y w h e n a s k e d to describe sensations o c c u r r i n g w i t h CO2 i n h a l a t i o n ( C a i n and M u r p h y ,  1980).  R a t s c a n detect CO2 at concentrations b e t w e e n 0 . 0 4 and 1 . 7 % ( Y o u n g e n t o b , 1991), w h i c h is far b e l o w the l e v e l r e q u i r e d to stimulate t r i g e m i n a l neurons ( P e p p e l a n d A n t o n , 1993). T h e exact quality o f C 0  2  that rats are p e r c e i v i n g at these l o w levels is u n k n o w n , but a large p o r t i o n o f the 15  rat b r a i n is devoted to odour detection so rats m a y b e m o r e sensitive to the odour  quality  associated w i t h CO2 than h u m a n s . W a l l a c e a n d R o s e n (2000) demonstrated that exposure o f rats to n o v e l odours, s u c h as b u t y r i c a c i d ( s i m i l a r to r a n c i d butter) a n d i s o a m y l acetate ( s i m i l a r to banana), causes a v o i d a n c e , reduces g r o o m i n g t i m e a n d increases f r e e z i n g t i m e , suggesting that n o v e l odours c a n e l i c i t fear i n rats. T h u s , CO2 m a y cause distress i n rats because it i s a c t i n g as a n o v e l s t i m u l u s that elicits fear.  1.4 Assessment of Distress During C 0 Euthanasia 2  In the assessment o f a n i m a l s ' subjective states it i s not p o s s i b l e to o b t a i n v e r b a l reports o f their e x p e r i e n c e , a n d it is therefore necessary to use other measures. T h e r e are t w o types o f measures that c a n be u s e d to assess whether a procedure s u c h as CO2 euthanasia causes p a i n a n d distress i n a n i m a l s : 1) p h y s i o l o g i c a l measures o f stress, a n d 2) b e h a v i o u r a l measures o f distress and a v e r s i o n .  1.4.1 Physiological assessment of distress T h e t e r m stress generally refers to a b i o l o g i c a l response to a n actual or p e r c e i v e d threat to homeostasis (e.g. M o b e r g , 2 0 0 0 ) , a n d i n v o l v e s b o t h b e h a v i o u r a l a n d p h y s i o l o g i c a l responses that act to m a i n t a i n homeostasis. P h y s i o l o g i c a l responses, s u c h as a c t i v a t i o n o f the s y m p a t h e t i c a d r e n e r g i c - m e d u l l a r y axis ( S A M ) a n d the h y p o t h a l a m i c - p i t u i t a r y - a d r e n a l axis ( H P A ) , o c c u r i n response to b o t h p h y s i o l o g i c a l a n d p s y c h o l o g i c a l stressors a n d serve to redirect b o d y systems t o w a r d s c o p i n g w i t h the stressor. A c t i v a t i o n o f the S A M axis occurs a l m o s t i m m e d i a t e l y i n response  to a stressor.  This  system  activates  the b o d y  b y releasing  epinephrine a n d  n o r e p i n e p h r i n e , i n c r e a s i n g glucose m e t a b o l i s m , respiration, heart rate a n d b l o o d pressure, a n d reducing  functions  that are n o t o f i m m e d i a t e necessity  16  (summarized  b y Toates,  1995).  A c t i v a t i o n o f the H P A axis results i n release o f c o r t i c o t r o p h i n - r e l e a s i n g h o r m o n e ( C R H ) other secretagogues f r o m the h y p o t h a l a m u s , that lead to the release o f  and  adrenocorticotropic  h o r m o n e ( A C T H ) f r o m the pituitary and then Cortisol or corticosterone ( i n the rat) f r o m the adrenal cortex. T h i s s y s t e m causes a n u m b e r o f effects i n c l u d i n g m o b i l i z a t i o n o f energy, s u p p r e s s i o n o f the i m m u n e s y s t e m , and release o f e n d o r p h i n s to m o d u l a t e p a i n responses ( s u m m a r i z e d b y Toates, 1995). T h e presence or absence o f a c t i v a t i o n o f the S A M and H P A axes c a n p r o v i d e i n f o r m a t i o n as to whether a s t i m u l u s acts as a stressor, but as w i l l be discussed later, their usefulness for assessing distress is l i m i t e d . C o n s i s t e n t a c t i v a t i o n o f the S A M a x i s i n response to r e d u c e d O2 a n d elevated CO2 has not been o b s e r v e d i n rats. B o r o v s k y et a l . (1998) f o u n d that a 3 0 s exposure to 1 0 0 % CO2 or 1 0 0 % n i t r o g e n w a s s u f f i c i e n t to increase p l a s m a norepinephrine levels i n rats, and F u k u d a et a l . (1989) f o u n d that 1 0 0 % CO2 increases cardiac sympathetic a c t i v i t y . H o w e v e r , other studies u s i n g l o w e r CO2 concentrations and higher O2 concentrations have f a i l e d to observe a c t i v a t i o n o f the S A M a x i s . H o d g e s et a l . (2002) f o u n d that w h e n rats were e x p o s e d to either 1 2 % O2 i n air or 7 % CO2 i n O2, v e n t i l a t i o n increased, but heart rate and b l o o d pressure d i d not. S i m i l a r l y , R a f f a n d R o a r t y (1988) f o u n d that b l o o d pressure w a s not affected b y l o w O2 (10%) O2), h i g h CO2 ( 4 % or 8 % C 0 ) or a c o m b i n a t i o n o f l o w 0 2  2  and h i g h C 0  2  (7% 0  2  and either 4 % or 8 % C 0 ) . 2  T h r e e studies have e x a m i n e d S A M variables d u r i n g CO2 euthanasia i n rats. W i t h p r e - f i l l e x p o s u r e , a l l studies f o u n d that heart rate ( C o e n e n et a l . , 1 9 9 5 ; Sharp et a l . , 2 0 0 6 ; S m i t h and Harrap,  1997)  and  blood  pressure  (Sharp,  2006;  Smith  and  Harrap,  1997)  decreased  i m m e d i a t e l y . W i t h g r a d u a l - f i l l exposure, C o e n e n et a l . (1995) f o u n d that heart rate decreased, but S m i t h and H a r r a p (1997) f o u n d that heart rate and b l o o d pressure i n i t i a l l y increased before d e c l i n i n g . T h e i m m e d i a t e d e c l i n e i n heart rate w i t h p r e - f i l l euthanasia c a n p r o b a b l y be e x p l a i n e d b y r a p i d depression o f the C N S . A l s o , irritant s t i m u l i , i n c l u d i n g CO2 at h i g h concentrations, are k n o w n to cause b r a d y c a r d i a v i a s t i m u l a t i o n o f t r i g e m i n a l nociceptors ( Y a v a r i et a l . , 1996). 17  Unfortunately the results for gradual fill are difficult to interpret because neither study used handled controls or acclimatized the animals to the chamber. It is therefore unclear how these variables relate to resting values, and whether activation of the S A M axis occurred as a result of handling. Increased H P A axis activity during moderate hypoxia and hypercapnia has also been examined in rats. Marotta et al. (1976) found that prolonged exposure to low O2 (10% O2 in nitrogen) or high CO2 (10% CO2, 20% O2, in nitrogen) resulted in corticosterone levels almost eight times those observed in control animals. Similarly, Raff and Roarty (1988) measured A C T H after exposure to low 0 (10% 0 ) , high C 0 (4% C 0 or 8% C 0 ) or a combination of 2  2  2  2  2  low 0 and high C 0 (7% 0 and either 4% or 8% C 0 ) , and found that it was elevated with 8% 2  2  2  2  CO2 and with the combination mixtures. Although one study has specifically examined A C T H and corticosterone levels during gradual-fill CO2 euthanasia and found no increase (Hackbarth et al., 1999), the results were likely affected by problems with experimental design. Not only were the sample sizes extremely small for analysis of these variables (N = 4), but decapitation was performed 30 s, 75 s, and 120 s into the procedure. This time frame does not allow for increases in these hormones to occur in the blood stream (Terlouw et al., 1997), thus no effect could be expected. The two previous studies indicate that gradual-fill CO2 euthanasia could result in activation of the HPA axis. There are, however, three major limitations with the use of physiological stress measures in the assessment of distress associated with CO2 euthanasia. Firstly, S A M and HPA activation do not imply that the stressor or the response is negative or positive for the animal. In fact, physiological stress responses are associated with neutral and positive events such as exercise and mating. Thus a stress response does not necessarily mean that an animal's welfare is compromised (Dawkins, 1998). However, by examining whether the animal finds the stressor  18  appetitive or a v e r s i v e , it is often p o s s i b l e to determine whether the stressor is p e r c e i v e d i n a negative or p o s i t i v e manner. Secondly,  S A M and H P A  a c t i v a t i o n o c c u r i n response to b o t h p h y s i o l o g i c a l  and  p s y c h o l o g i c a l stressors, s u c h that the actual effects o f the s t i m u l u s o n the subjective state o f the a n i m a l are d i f f i c u l t to determine. In the case o f the H P A a x i s , it appears that there are at least t w o types o f C N S pathways that c a n e l i c i t release o f C R H . H e r m a n and C u l l i n a n (1997) suggest that there are direct p a t h w a y s for i m m e d i a t e p h y s i o l o g i c a l threats, and indirect p a t h w a y s v i a higher b r a i n c i r c u i t s for s t i m u l i that require interpretation w i t h respect to p r e v i o u s experience. H P A responses to what the authors l a b e l " p r o c e s s i v e " stressors l i k e restraint and n o v e l t y appear to be associated w i t h relay t h r o u g h higher b r a i n areas s u c h as the f o r e b r a i n and l i m b i c system n u c l e i , whereas responses to " s y s t e m i c " stressors l i k e h y p o x i a and ether exposure are m o r e l i k e l y d i r e c t l y r e l a y e d f r o m brainstem n u c l e i ( r e v i e w e d b y H e r m a n a n d C u l l i n a n , 1 9 9 7 ; H e r m a n et a l . , 1996). T h i s suggests that H P A  responses to CO2 euthanasia c o u l d o c c u r  without  p e r c e p t i o n b y higher b r a i n centres. I f so, they w o u l d be a p o o r measure o f p s y c h o l o g i c a l distress associated w i t h this procedure. In fact, it has been s h o w n that h y p o t h a l a m i c  vasopressin-  c o n t a i n i n g neurons, w h i c h secrete the C R H secretogogue v a s o p r e s s i n , p r o d u c e s i m i l a r increases i n c - F o s ( i n d i c a t i n g a c t i v a t i o n o f the neuron) i n response to CO2 exposure i n b o t h a w a k e and anesthetized rats ( K c et a l . , 2 0 0 2 ) . A l t h o u g h these measures c a n p r o v i d e a gauge o f h o w the s t i m u l u s affects the a n i m a l p h y s i o l o g i c a l l y , and thus the potential for c a u s i n g distress, they do not n e c e s s a r i l y a l l o w one to assess the p s y c h o l o g i c a l state o f the a n i m a l . F i n a l l y , because CO2 euthanasia occurs r e l a t i v e l y q u i c k l y , it is not p o s s i b l e to measure H P A responses to the p o r t i o n o f the procedure that occurs w h i l e the a n i m a l is still c o n s c i o u s . In p r e - f i l l and gradual CO2 euthanasia, rats b e c o m e u n c o n s c i o u s i n a p p r o x i m a t e l y 15 s and 90 s r e s p e c t i v e l y , and i n terms o f distress w e are c o n c e r n e d o n l y w i t h what the a n i m a l experiences d u r i n g this t i m e . C h a n g e s i n the S A M o c c u r w i t h i n seconds a n d c a n be measured d u r i n g this 19  p r o c e d u r e , but e v i d e n c e o f a c t i v a t i o n o f the H P A a x i s does not appear this q u i c k l y and the state o f the a n i m a l at the t i m e o f loss o f consciousness cannot be preserved. B y o b s e r v i n g this system after p r o l o n g e d exposure or after the a n i m a l regains c o n s c i o u s n e s s , w e are n o longer o b s e r v i n g w h a t o c c u r r e d d u r i n g that l i m i t e d p e r i o d o f t i m e . F o r e x a m p l e , n o c i c e p t i v e CO2 concentrations m i g h t o c c u r after loss o f c o n s c i o u s n e s s , and this m i g h t have a n effect o n H P A variables that are not associated w i t h the a n i m a l ' s c o n s c i o u s experience.  1.4.2 Behavioural assessment of distress U n l i k e p h y s i o l o g i c a l measures, b e h a v i o u r a l measures c a n be e a s i l y m o n i t o r e d d u r i n g the course o f CO2 exposure, and the types o f b e h a v i o u r s p e r f o r m e d c a n p r o v i d e us w i t h i n f o r m a t i o n about h o w the a n i m a l perceives the s t i m u l u s . D u r i n g the euthanasia process w e c a n observe w h e t h e r the a n i m a l e x h i b i t s b e h a v i o u r s that are i n d i c a t i v e o f distress s u c h as escape b e h a v i o u r s , distress v o c a l i z a t i o n s and b e h a v i o u r s associated w i t h p a i n or d i s c o m f o r t . In tests o f a v e r s i o n w e c a n observe whether the a n i m a l a v o i d s CO2 exposure, and whether it w i l l p a y a cost to a v o i d exposure. W e c a n also use other b e h a v i o u r a l tests to determine whether CO2 exposure elicits fear i n a n i m a l s .  1.4.2.1 Behaviour during CO2 exposure D u r i n g exposure to a n aversive or p a i n f u l gas, a n i m a l s m i g h t s h o w b o t h general and s p e c i e s - s p e c i f i c signs o f distress. G e n e r a l signs m i g h t i n c l u d e b e h a v i o u r s associated w i t h escape, s u c h as increased a c t i v i t y , e x p l o r a t i o n , and concentrated a c t i v i t y at k n o w n exits. In the rat this m i g h t be i n d i c a t e d b y increased l o c o m o t o r a c t i v i t y and r e a r i n g , as w e l l as w a l l - c l i m b i n g , i n v e s t i g a t i o n o f c h a m b e r surfaces and p u s h i n g , s c r a t c h i n g and b i t i n g at potential exits. A l t h o u g h a n increase i n a c t i v i t y indicates that the a n i m a l is r e s p o n d i n g to the s t i m u l u s , a n increase i n actual escape b e h a v i o u r s , s u c h as p u s h i n g , s c r a t c h i n g and b i t i n g at potential e x i t s , is important 20  to s h o w that the s t i m u l u s is p e r c e i v e d as negative and is not s i m p l y e l i c i t i n g e x p l o r a t i o n . R a t s have also b e e n s h o w n to r e s p o n d to aversive s t i m u l i b y freezing (e.g. Barnett, 1 9 7 5 ; D i e l e n b e r g and M c G r e g o r , 2 0 0 1 ) , w h i c h differs f r o m s i m p l e i n a c t i v i t y i n terms o f increased m u s c l e tone a n d a l a c k o f head m o v e m e n t s . A l t h o u g h this seems contradictory to the increase i n a c t i v i t y d e s c r i b e d a b o v e , it is w i d e l y r e c o g n i z e d that a n i m a l s , i n c l u d i n g rats, c a n r e s p o n d p r o a c t i v e l y or r e a c t i v e l y w h e n c o n f r o n t e d w i t h a stressor ( r e v i e w e d b y K o o l h a s et a l . , 1999).  Therefore  distress c o u l d be i n d i c a t e d b y either a n increase i n a c t i v i t y a n d escape b e h a v i o u r or b y f r e e z i n g b e h a v i o u r . In assessment o f either b e h a v i o u r a l response, it is important to c o m p a r e it to stable baseline or c o n t r o l data to ensure that the response is i n fact due to s t i m u l u s exposure, and not to s o m e other factor s u c h as h a n d l i n g or exposure to a n o v e l e n v i r o n m e n t . D i s t r e s s c a n also be i n d i c a t e d b y the p r o d u c t i o n o f v o c a l i z a t i o n s . V o c a l i z a t i o n s p r o d u c e d d u r i n g distress m i g h t serve a n u m b e r o f purposes, s u c h as to w a r n c o n s p e c i f i c s o f danger, to e l i c i t a c a r e - g i v i n g response or to assist i n r e g a i n i n g contact w i t h c o n s p e c i f i c s ( r e v i e w e d  by  K l u m p a n d Shalter, 1984, r e p r o d u c e d i n H a u s e r , 1996). R a t s have b e e n s h o w n to produce u l t r a s o n i c v o c a l i z a t i o n s ( U S V s ) i n a variety o f contexts, and these c a l l s m a y be an i n d i c a t o r o f a f f e c t i v e state ( K n u t s o n et a l . , 2 0 0 2 ) . U S V s i n the 50 k H z a n d 2 0 k H z ranges tend to o c c u r i n response to appetitive and aversive s t i m u l i respectively. F o r e x a m p l e , U S V s i n the 2 0 - 3 0 k H z range have been o b s e r v e d i n response to p a i n f u l s t i m u l a t i o n ( D i n h et a l . , 1 9 9 9 ; C o l p a e r t et a l . , 1 9 8 7 ; J o u r d a n et a l . , 1 9 9 5 , 1998), exposure to predators ( B l a n c h a r d et a l . , 1991), w i t h d r a w a l ( V i v i a n a n d M i c z e k , 1991), f i g h t i n g w i t h c o n s p e c i f i c s ( T h o m a s  drug  et a l . , 1983),  acoustic startle ( K a l t w a s s e r , 1990) active a v o i d a n c e l e a r n i n g ( C u o m o et a l . , 1992) and t o u c h i n g by an unfamiliar human (Brudzynski  and O c i e p a , 1992). W h i l e these c a l l s appear to be  r e l a t i v e l y n o n - s p e c i f i c a n d their s i g n a l l i n g context is not f u l l y u n d e r s t o o d , they d o have an a s s o c i a t i o n w i t h situations that appear to be negative.  21  T h e occurrence o f b e h a v i o u r s associated w i t h p a i n or w i t h a c t i v a t i o n o f a stress response m a y a l s o indicate that CO2 euthanasia causes distress. P a i n - r e l a t e d b e h a v i o u r s m i g h t i n c l u d e s h a k i n g o f the head or p a w i n g at the nose and eyes. U r i n a t i o n and d e f e c a t i o n are also sometimes used as measures o f distress because they are associated w i t h a c t i v a t i o n o f the a u t o n o m i c nervous s y s t e m d u r i n g the stress response ( r e v i e w e d b y V i n g e r h o e t s et a l . , 1985). A l t h o u g h u r i n a t i o n a n d d e f e c a t i o n c a n be indicators o f distress, their absence does not necessarily indicate a l a c k o f distress because they c a n be affected b y recent f e e d i n g and e l i m i n a t i o n behaviour. A n u m b e r o f studies have o b s e r v e d the b e h a v i o u r o f rats d u r i n g CO2 euthanasia, but often c o n c l u s i o n s are d r a w n o n the basis o f o p i n i o n rather than objective data. R e s p o n s e s to CO2 have been characterized b y some as " a p p r e h e n s i v e " (Hornett and H a y n e s , 1984), or " a b n o r m a l " , " e x c i t e d " a n d " a g i t a t e d " ( C o e n e n et a l . , 1995), w i t h o u t precise d e f i n i t i o n s or descriptions o f b e h a v i o u r a l measures. H a c k b a r t h et a l . (1999) suggest that they s a w no signs o f " f e a r " i n rats d u r i n g CO2 euthanasia. H o w e v e r , no data are p r o v i d e d to support this assertion, even t h o u g h they describe a n u m b e r o f b e h a v i o u r a l variables that were m e a s u r e d . O t h e r studies have used objective measures o f b e h a v i o u r , but the usefulness o f these measures is c o m p l i c a t e d b y s m a l l sample sizes or a l a c k o f appropriate distress criteria, controls or a c c l i m a t i z a t i o n p r i o r to gas exposure. B l a c k s h a w et a l . (1988) c o m p a r e d rats' responses d u r i n g exposure to either air or p r e - f i l l CO2, and measured m o v e m e n t , t i m e spent stationary and w a l l touches and c l i m b s d u r i n g the first 10 s o f exposure. D u r i n g CO2 exposure, rats e x h i b i t e d less o v e r a l l a c t i v i t y , but m o r e w a l l c l i m b i n g . T h e decrease i n a c t i v i t y is d i f f i c u l t to interpret because it m a y be due to the a n i m a l f r e e z i n g or the anaesthetic effects o f CO2. H o w e v e r , the increased i n c i d e n c e o f w a l l c l i m b i n g does suggest increased e x p l o r a t i o n . W a l l c l i m b i n g w a s e v e n greater d u r i n g exposure to ether and c h l o r o f o r m , w h i c h are k n o w n irritants, suggesting that this b e h a v i o u r w a s i n d i c a t i v e o f s o m e l e v e l o f distress, but also that the response w a s not  22  m a x i m a l . H o w e v e r , the results o f this study m u s t be interpreted w i t h c a u t i o n because o n l y three a n i m a l s per g r o u p were observed. S m i t h and H a r r a p (1997) recorded the b e h a v i o u r a l responses o f rats to b o t h r a p i d - f i l l and g r a d u a l - f i l l euthanasia. T h e y observed w a l l c l i m b i n g i n o n l y one a n i m a l a n d d i d not observe any other escape b e h a v i o u r s , but they d i d observe c i r c l i n g o f the c h a m b e r b y the m a j o r i t y o f a n i m a l s i n b o t h groups and i m m e d i a t e u r i n a t i o n b y a l l rats i n the r a p i d fill group. T h e c i r c l i n g b e h a v i o u r w a s not q u a n t i f i e d so it is not k n o w n whether it d i f f e r e d b e t w e e n groups, and this study d i d not use controls or a c c l i m a t i z a t i o n , so it i s unclear h o w m u c h o f the effect w a s due to h a n d l i n g and n o v e l t y . H o w e v e r , u r i n a t i o n b y a l l rats i n one g r o u p and none i n the other does suggest distress i n the r a p i d f i l l group. F i n a l l y , B r i t t (1987) e x a m i n e d the responses o f t w o strains o f rats d u r i n g baseline and d u r i n g exposure to either p r e - f i l l o r g r a d u a l - f i l l CO2 euthanasia. H o w e v e r , interpretation o f this data is d i f f i c u l t because statistical analyses were not p e r f o r m e d and i n f o r m a t i o n o n v a r i a b i l i t y w a s not p r o v i d e d . P r e - f i l l euthanasia w a s o n l y c o m p l e t e d w i t h one rat. D u r i n g  gradual-fill  e x p o s u r e , Sprague D a w l e y rats s h o w e d increases i n activity and c l i m b i n g , w h i l e L i s t e r H o o d e d rats s h o w e d a decrease i n a c t i v i t y , n o change i n c l i m b i n g a n d a n increase i n  backward  m o v e m e n t s . Increased r e a r i n g and m o v i n g indicate increased e x p l o r a t i o n , a n d the r e d u c t i o n i n a c t i v i t y i n the second strain i s ' d i f f i c u l t to interpret because o f the n a r c o t i c effects o f CO2. T h e presence o f b a c k w a r d m o v e m e n t s , w h i c h are not generally seen i n rats, m a y indicate that the a n i m a l s were t r y i n g to r e m o v e themselves from the s t i m u l u s . B o t h strains also s h o w e d increases i n s h a k i n g , u r i n a t i o n a n d defecation. Increased s h a k i n g indicates that contrary to suggestions,  g r a d u a l - f i l l exposure  increased u r i n a t i o n  previous  m a y result i n p a i n p r i o r to loss o f c o n s c i o u s n e s s ,  and defecation suggest  a c t i v a t i o n o f the  autonomic  nervous  H o w e v e r , s h a k i n g w a s not d e f i n e d , m a k i n g the o c c u r r e n c e o f this b e h a v i o u r  and  system.  d i f f i c u l t to  23  \  interpret. A l t h o u g h u l t r a s o n i c data were r e c o r d e d , no v o c a l i z a t i o n s were detected for either strain. In s u m m a r y , the m a j o r i t y o f research o n the b e h a v i o u r a l responses o f rats to  CO2  exposure has b e e n p o o r l y d e s i g n e d a n d the results are d i f f i c u l t to interpret. W h i l e some studies have f o u n d n o e v i d e n c e o f distress d u r i n g C O 2 exposure, others h a v e f o u n d b e h a v i o u r s that suggest distress a n d p o s s i b l y p a i n . W e l l - c o n t r o l l e d research is necessary to c l a r i f y these discrepancies.  1.4.2.2 Aversion  Testing  A v e r s i o n testing c a n be u s e d to e x a m i n e whether a n a n i m a l w i l l a v o i d a s t i m u l u s , as w e l l as the strength o f a v e r s i o n to that s t i m u l u s . F i r s t l y , w e c a n observe whether a n a n i m a l w i l l r e m o v e i t s e l f f r o m a s t i m u l u s i f g i v e n the o p p o r t u n i t y , and h o w q u i c k l y it does so. S e c o n d l y , w e c a n test the strength o f a v e r s i o n to the s t i m u l u s b y h a v i n g the a n i m a l w o r k to a v o i d the s t i m u l u s , or b y c o m p a r i n g the s t i m u l u s to another s t i m u l u s o f k n o w n v a l u e u s i n g either a p p r o a c h a v o i d a n c e testing or a v o i d a n c e - a v o i d a n c e testing. In a p p r o a c h - a v o i d a n c e c o n f l i c t , a n attractive s t i m u l u s is p a i r e d w i t h an aversive s t i m u l u s , and the a n i m a l m u s t determine whether it is w i l l i n g to accept the aversive s t i m u l u s i n order to g a i n access to the attractive one. In a v o i d a n c e a v o i d a n c e testing, the a n i m a l m u s t choose b e t w e e n exposure to one o f t w o aversive s t i m u l i . B y c o m p a r i n g the test s t i m u l u s to other s t i m u l i o f k n o w n v a l u e , it is p o s s i b l e to rate h o w aversive the test s t i m u l u s is ( R u s h e n , 1996). 'A  Preference  studies  have  been  used  to  determine  whether  rats  find  different  concentrations o f C O 2 aversive, and h o w these rank against a r g o n - i n d u c e d h y p o x i a ( < 2 % O 2 ) a n d gaseous anaesthetics. L e a c h et a l . (2002 a,b) tested rats i n a preference system c o n s i s t i n g o f t w o c h a m b e r s connected b y a t u n n e l , where one c h a m b e r c o n t a i n e d the test gas and the other c o n t a i n e d air. D u r i n g c o n t r o l sessions w i t h air, rats w i t h d r e w 24  f r o m the test c h a m b e r  in  a p p r o x i m a t e l y 15 s, whereas d u r i n g testing w i t h CO2 at concentrations greater than 2 5 . 5 % , rats w i t h d r e w f r o m the test c h a m b e r i n approxi m atel y. 1 s. F u r t h e r m o r e , d u r i n g the 180 s test session, the t i m e spent i n the CO2 c h a m b e r w a s o n l y 1 to 2 s, w h i l e the t i m e spent i n this c h a m b e r d u r i n g c o n t r o l sessions w a s 35 to 50 s. T i m e to exit a n d t i m e spent i n the c h a m b e r were s i g n i f i c a n t l y greater f o r severe h y p o x i a and anaesthetic gases, suggesting that h y p o x i a and anaesthetic gases are less aversive than CO2. These results indicate that rats are able to q u i c k l y detect CO2 at concentrations greater t h a n 2 5 . 5 % a n d that they f i n d these concentrations aversive.  However,  because there w a s no cost to l e a v i n g the c h a m b e r , these results do not indicate the strength o f a v e r s i o n to CO2.  Furthermore,  these results o n l y  s h o w the response to moderate,  static  concentrations o f CO2, and responses to g r a d u a l - f i l l exposure m i g h t d i f f e r . It is thought that anaesthesia begins to o c c u r at l o w levels o f CO2, and a n i m a l s m a y therefore be p a r t i a l l y anesthetized w h e n moderate concentrations o f CO2 o c c u r d u r i n g g r a d u a l - f i l l exposure. W h i l e the strength o f a v e r s i o n to CO2 has not yet been e x a m i n e d i n rats, a p p r o a c h a v o i d a n c e testing has been used to e x a m i n e CO2 a v e r s i o n i n other species. Studies w i t h p o u l t r y have  found  that  a large  proportion  of  birds  will  enter  a chamber  containing  CO2  at  concentrations greater than 6 0 % i n order to g a i n access to f o o d or s o c i a l contact ( G e r r i t z e n , et a l . , 2 0 0 0 ; R a j , 1 9 9 6 ; W e b s t e r & F l e t c h e r , 2 0 0 4 ) . Interestingly, R a j (1996) f o u n d that birds that entered a c h a m b e r c o n t a i n i n g 7 5 % CO2 d i d so w h i l e e x h i b i t i n g s y m p t o m s o f p a i n and distress s u c h as g a s p i n g , h e a d s h a k i n g and v o c a l i z i n g . A v e r s i o n to CO2 has also been e x a m i n e d i n p i g s , a n d w h i l e they w i l l generally tolerate 3 0 % CO2 i n order to g a i n access to a f o o d r e w a r d , they w i l l not tolerate exposure to 9 0 % CO2, e v e n after a 2 4 - h p e r i o d o f f o o d d e p r i v a t i o n (Raj and G r e g o r y , 1995). M i n k have been s h o w n to w o r k to obtain access to n o v e l objects ( M a s o n et a l . , 2 0 0 1 ) , but w i l l a v o i d a c h a m b e r c o n t a i n i n g a n o v e l object w h e n it contains 1 0 0 % CO2 ( C o o p e r et a l . , 1998). T h e fact that p i g s and m i n k are w i l l i n g to f o r g o access to desired items to a v o i d h i g h concentrations o f CO2 suggests that it is aversive. H o w e v e r , 25  responses to moderate  CO2  concentrations that are s u f f i c i e n t to i n d u c e loss o f consciousness have not been e x a m i n e d . S i m i l a r studies are needed to determine whether rats e x h i b i t s i g n i f i c a n t a v e r s i o n to  CO2  exposure.  1.4.2.3 Tests ofanxiety' Researchers have used the V o g e l c o n f l i c t test to determine whether CO2 causes what they have t e r m e d ' a n x i e t y ' i n rats. F o r the V o g e l c o n f l i c t test, rats are g i v e n l i m i t e d access to water, and d u r i n g s p e c i f i e d periods they c a n d r i n k but o n l y w i t h c o n c o m i t a n t exposure to m i l d electric s h o c k . It is p r e d i c t e d that w h e n an a n x i e t y - p r o v o k i n g s t i m u l u s is d e l i v e r e d p r i o r to water access, d r i n k i n g w i l l be r e d u c e d : C u c c h e d d u et a l . (1995) o b s e r v e d the effect o f a 1 0 m i n u t e exposure to gradual fill w i t h a 3 5 % CO2 / 6 5 % O2 m i x t u r e , and f o u n d a 4 0 % r e d u c t i o n i n d r i n k i n g . T h i s result w a s s i m i l a r to the effect o f d o s i n g w i t h an a n x i o g e n i c c o m p o u n d . It w a s also d e t e r m i n e d that this s u p p r e s s i o n c o u l d be e l i m i n a t e d b y d o s i n g the rats w i t h a n x i o l y t i c s p r i o r to CO2 exposure. A l t h o u g h this study uses a n indirect m e t h o d o f measurement, it does indicate that exposure to a n i n c r e a s i n g c o n c e n t r a t i o n o f CO2 causes a n x i e t y i n rats.  1.4.2.4 Summary of Behavioural Measures: M o s t studies have not f o u n d b e h a v i o u r s that are i n d i c a t i v e o f distress d u r i n g exposure o f rats to CO2, either u s i n g the p r e - f i l l or g r a d u a l - f i l l methods. H o w e v e r , the results o f b e h a v i o u r a l tests w i t h rats suggest that they are averse to concentrations o f CO2 that are sufficient to cause loss o f c o n s c i o u s n e s s . A v e r s i o n to g r a d u a l - f i l l CO2 exposure has not yet been e x a m i n e d , and the strength o f CO2 a v e r s i o n i n rats is s t i l l u n k n o w n .  26  1.5 Objectives A l t h o u g h p r e - f i l l CO2 euthanasia o f rats is r e l a t i v e l y fast, h u m a n self-report data a n d data o n n o c i c e p t o r s t i m u l a t i o n suggest that it has a h i g h potential for p a i n i n rats. It also has a h i g h potential for n o n - p a i n distress due to dyspnea. T h e potential for distress d u r i n g g r a d u a l - f i l l CO2 euthanasia is less clear because loss o f consciousness occurs at a p p r o x i m a t e l y 4 0 % CO2 ( S m i t h a n d H a r r a p , 1997). In general, 4 0 % CO2 is not s u f f i c i e n t to cause p a i n i n h u m a n s , or to stimulate nociceptors i n rat nasal m u c o s a . H o w e v e r , it is s u f f i c i e n t to cause d y s p n e a i n h u m a n s , and it is p o s s i b l e that rats also experience this sensation d u r i n g exposure to increased levels o f CO2. T h e m a j o r i t y o f studies e x a m i n i n g responses to CO2 exposure have not observed clear b e h a v i o u r a l signs o f distress i n rats, but the studies to date have been p o o r l y  executed.  B e h a v i o u r a l testing has d e t e r m i n e d that CO2 exposure causes a v e r s i o n i n rats a n d other species, but the strength o f rats' a v e r s i o n to CO2 and the effects o f g r a d u a l - f i l l exposure have not yet been d e t e r m i n e d . F r o m the i n f o r m a t i o n currently a v a i l a b l e it is not p o s s i b l e to determine c o n c l u s i v e l y whether either m e t h o d o f CO2 euthanasia causes distress i n rats. H o w e v e r , p r e - f i l l CO2 exposure has a h i g h potential for c a u s i n g p a i n , so I d e c i d e d to f o c u s m y dissertation research o n g r a d u a l fill  CO2 euthanasia. T h e t w o m a i n objectives o f m y thesis w e r e :  g r a d u a l - f i l l CO2 responses  during  euthanasia causes distress i n laboratory euthanasia, a n d a v e r s i o n d u r i n g  rats, b y  1) to determine whether examining  approach-avoidance  behavioural  testing, a n d 2)  to  determine whether p a i n , d y s p n e a and n o v e l t y are l i k e l y sources o f distress d u r i n g g r a d u a l - f i l l CO2 euthanasia. In m y first study ( C h a p t e r 2) I p e r f o r m e d a detailed analysis o f b e h a v i o u r a l responses o f rats d u r i n g g r a d u a l - f i l l CO2 euthanasia i n order to determine whether they s h o w b e h a v i o u r a l signs o f distress. I also e x a m i n e d rats' responses to a r e d u c t i o n i n O2 concentrations  27  to  determine whether h y p o x i a p l a y e d a role i n their responses to CO2. In m y s e c o n d study (Chapter 3), I u s e d a p p r o a c h - a v o i d a n c e testing to determine whether rats f i n d CO2 m o r e aversive than a v a l u a b l e f o o d r e w a r d , and to determine w h i c h concentrations o f CO2 rats f i n d aversive w h e n tested w i t h either static or g r a d u a l l y i n c r e a s i n g CO2. I also e x a m i n e d rat a v e r s i o n to a r g o n i n d u c e d h y p o x i a , w h i c h has been suggested as an alternative gas euthanasia m e t h o d . In m y t h i r d study  (Chapter 4 ) , I e x p a n d e d m y  i n v e s t i g a t i o n o f rat a v e r s i o n to g r a d u a l l y  concentrations o f CO2 b y d e t e r m i n i n g whether C 0  2  increasing  flow rate affects rat a v e r s i o n to CO2. W i t h i n  the first three studies I also e x a m i n e d the CO2 concentrations that resulted i n b e h a v i o u r a l signs o f distress and a v e r s i o n , and c o m p a r e d these values w i t h those f r o m p r e v i o u s studies o n the p o t e n t i a l for CO2 to cause p a i n and d y s p n e a . In d o i n g this I w a s able to assess whether p a i n and d y s p n e a were l i k e l y causes o f this, distress and a v e r s i o n . In m y  final  study (Chapter 5), I  investigated whether n o v e l t y w a s a cause o f distress and a v e r s i o n d u r i n g g r a d u a l - f i l l CO2 exposure.  1)  28  1.6 References 2 0 0 0 . T h e C a n a d i a n O x f o r d D i c t i o n a r y , B i s s e t , A . (ed). D o n M i l l s : O x f o r d U n i v e r s i t y Press. A m b r o s e , N . , W a d h a m , J . , M o r t o n , D . , 2 0 0 0 . R e f i n e m e n t i n E u t h a n a s i a . In: B a l l s , M . , v a n Z e l l e r , A . M . , H a i d e r , M . E . ( E d s ) , Progress i n the R e d u c t i o n , R e f i n e m e n t and R e p l a c e m e n t of A n i m a l Experimentation, Elsevier Science, Amsterdam, pp.1159-1169. A m e r i c a n Veterinary  M e d i c a l Association, 2001. 2000 Report  o f the A V M A  Panel  on  E u t h a n a s i a . J o u r n a l o f the V e t e r i n a r y M e d i c a l A s s o c i a t i o n 2 1 8 , 6 6 9 - 6 9 6 . A n t o n , F., E u c h n e r , I., H a n d w e r k e r , H . O . ,  1992. Psychophysical examination o f pain induced  b y d e f i n e d CO2 pulses a p p l i e d to the nasal m u c o s a . P a i n 4 9 , 5 3 - 6 0 . A n t o n , F., P e p p e l , P., E u c h n e r , I.,  Handwerker, H.O.,  1991. Controlled noxious chemical  s t i m u l a t i o n : responses o f rat t r i g e m i n a l b r a i n s t e m neurones to CO2 pulses a p p l i e d to the nasal m u c o s a . N e u r o s c i . Lett. 1 2 3 , 2 0 8 - 2 1 1 . A u s t r a l i a n and N e w Z e a l a n d C o u n c i l for the C a r e o f A n i m a l s i n R e s e a r c h and T e a c h i n g , 1 9 9 3 . E u t h a n a s i a o f A n i m a l s U s e d for S c i e n t i f i c P u r p o s e s , A N Z C C A R T , G l e n O s m o n d . B a n z e t t , R . B . , L a n s i n g , R . W . , E v a n s , K . C . , S h e a , S . A . , 1996. S t i m u l u s - r e s p o n s e characteristics o f C 0 2 - i n d u c e d air hunger i n n o r m a l subjects. R e s p . P h y s i o l . 1 0 3 , 1 9 - 3 1 . Barnett, S . A . 1 9 7 5 . T h e R a t : A S t u d y i n B e h a v i o r . C h i c a g o : T h e U n i v e r s i t y o f C h i c a g o Press. Bergersweeney, J., Berger, U.V.,  Sharma, M . , Paul, C.A.,  1994. E f f e c t s o f c a r b o n d i o x i d e -  i n d u c e d anaesthesia o n c h o l i n e r g i c parameters i n r a t - b r a i n . L a b . A n i m . S c i . 4 4 , 3 6 9 - 3 7 1 . B l a c k m o r e , D . K . , 1 9 9 3 . E u t h a n a s i a ; not a l w a y s eu. A u s t . V e t . J . 7 0 , 4 0 9 - 4 1 3 . B l a c k s h a w , J . K . , F e n w i c k , D . C . , B e a t t i e , A . W . , A l l a n , D . J . , 1988. T h e b e h a v i o u r o f c h i c k e n s , m i c e and rats d u r i n g euthanasia w i t h c h l o r o f o r m , c a r b o n d i o x i d e a n d ether. L a b . A n i m . 2 2 , 67-75. B l a n c h a r d , R . J . , B l a n c h a r d , D . C . , A g u l l a n a , R., W e i s s , S . M . , 1 9 9 1 . T w e n t y - t w o k H z a l a r m cries  29  to presentation o f a predator, b y laboratory rats l i v i n g i n v i s i b l e b u r r o w systems. P h y s i o l . Behav. 50, 967-972. B l o c k , N . , 1 9 9 5 . O n a c o n f u s i o n about a f u n c t i o n o f c o n s c i o u s n e s s . B e h a v . B r a i n S c i . 18, 2 2 7 287. B o r o v s k y , V . , H e r m a n , M . , D u n p h y , G . , C a p l e a , A . , E l y , D . , 1 9 9 8 . CO2 a s p h y x i a increases p l a s m a n o r e p i n e p h r i n e i n rats v i a sympathetic nerves. A m . J . o f P h y s i o l . 2 7 4 , R 1 9 - R 2 2 . B r i t t , D . P., 1 9 8 7 . T h e humaneness o f c a r b o n d i o x i d e as a n agent o f euthanasia for laboratory rodents. In: E u t h a n a s i a o f U n w a n t e d , Injured or D i s e a s e d A n i m a l s or f o r E d u c a t i o n a l or S c i e n t i f i c P u r p o s e s , U n i v e r s i t i e s F e d e r a t i o n for A n i m a l W e l f a r e , Potters B a r , p p . 1 9 - 3 1 . B r o d i e , D . A . , W o o d b u r y , D . M . , 1 9 5 8 . A c i d - b a s e changes i n b r a i n a n d b l o o d o f rats e x p o s e d to h i g h concentrations o f c a r b o n d i o x i d e . A m . J . P h y s i o l . 1 9 2 , 9 1 - 9 4 . B r u d z y n s k i , S . M . , O c i e p a , D . , 1992. U l t r a s o n i c v o c a l i z a t i o n o f laboratory rats i n response to h a n d l i n g and t o u c h . P h y s i o l . B e h a v . 5 2 , 6 5 5 - 6 6 0 . C a b l e , G . G . , 2 0 0 3 . I n - f l i g h t h y p o x i a incidents i n m i l i t a r y aircraft: causes and i m p l i c a t i o n s for t r a i n i n g . A v i a t . Space E n v i r o n . M e d . 7 4 , 1 6 9 - 1 7 2 . C a i n , W . S . , M u r p h y , C . L . , 1 9 8 0 . Interaction between c h e m o r e c e p t i v e m o d a l i t i e s o f o d o u r and irritation. N a t u r e 2 8 4 , 2 5 5 - 2 5 7 . C a n a d i a n C o u n c i l o n A n i m a l C a r e , 1 9 8 9 . E t h i c s o f A n i m a l Investigation (1989). A v a i l a b l e at: http://www.ccac.ca/en/CCAC  Programs/Guidelines  Policies/POLICIES/ETHICS.HTM.  Accessed M a y 2006. C a n a d i a n C o u n c i l o n A n i m a l C a r e , 1993. G u i d e to the C a r e and U s e o f E x p e r i m e n t a l A n i m a l s , V o l u m e 1, 2  n d  E d i t i o n , eds E . D . O l f e r t , B . M . C r o s s and A . A . M c W i l l i a m . O t t a w a , C C A C .  C a n a d i a n C o u n c i l o n A n i m a l C a r e , 2 0 0 6 . C C A C S u r v e y o f A n i m a l U s e - 2 0 0 4 . A v a i l a b l e at: http://www.ccac.ca/en/Publications/New  F a c t s Figures/analysis/analysis  Accessed M a y 2006. 30  index.htm.  Chen, X . , Gallar, J . , Pozo, M . A . , Baeza, M . , Belmonte, C , 1995. CO2 stimulation of the cornea: a comparison between human sensation and nerve  activity  in polymodal  nociceptive  afferents of the cat. Eur. J. Neurosci. 7, 1154-1163. Close, B . , Banister, K . , Baumans, V . , Bernoth, E . , Bromage, N . , Bunyan, J . , Erhardt, W . , Flecknell, P., Gregory, N . , Hackbarth, H . , Morton, D . , Warwick, C , 1997. Commission Working Party Report: Recommendations  for euthanasia  of  European  experimental  animals, Part I. Lab. A n i m . 30, 293-316. Coenen, A . M . , Drinkenburg, W . H . , Hoenderken, R., van Luijtelaar, G . L . , 1995. Carbon dioxide euthanasia in rats: oxygen supplementation minimizes signs of agitation and asphyxia. Lab. A n i m . 29, 262-268. Coenen, A . , Smit, A . , Zhonghua, L . , van Luijtelaar, G . , 2000. Gas mixtures for anaesthesia and euthanasia in broiler chickens. World Poultry Sci. J. 56, 225-234. Colpaert, F . C . , 1987. Evidence that adjuvant arthritis in the rat is associated with chronic pain. Pain 28, 201-222. Cooper, J . , Mason, G . , Raj, M . , 1998. Determination of the aversion of farmed mink (Mustela vison) to carbon dioxide. Vet. Rec. 143, 359-61. Cuccheddu, T., Floris, S., Serra, M . , Porceddu, M . L . , Sanna, E . , Biggio, G . , 1995. Proconflict effect of carbon dioxide inhalation in rats. Life Sci. 56, 321-324. Cuomo, V . , Cagiano, R., De Salvia, M . A . , Mazzoccoli, M . , Persichella, M . , Renna, G . , 1992. Ultrasonic vocalization as an indicator of emotional state during active avoidance learning in rats. Life Sci. 50, 1049-1055. Danneman, P.J., Stein, S., Walshaw, S.O., 1997. Humane and practical implications of using carbon dioxide mixed with oxygen for anaesthesia or euthanasia of rats. Lab. A n i m . Sci. 47, 376-85. Dawkins, M . S . , 1980. Animal Suffering: The Science of Animal Welfare. Chapman and Hall, 31  London. D a w k i n s , M . S . 1998. E v o l u t i o n and a n i m a l w e l f a r e . Q . R e v . B i o l . 7 3 : 3 0 5 - 3 2 8 . Dielenberg, R.A.,  Carrive,  P., M c G r e g o r ,  I.S.,  2001. The  c a r d i o v a s c u l a r and  behavioural  response to cat o d o r i n rats: u n c o n d i t i o n e d and c o n d i t i o n e d effects. B r a i n R e s . 8 9 7 , 2 2 8 - 2 3 7 . D i n h , H . K . , L a r k i n , A . , G a t l i n , L., P i e p m e i e r , E. Jr., 1 9 9 9 . R a t u l t r a s o u n d m o d e l for m e a s u r i n g p a i n r e s u l t i n g f r o m i n t r a m u s c u l a r l y injected a n t i m i c r o b i a l s . P D A J . P h a r m . S c i . T e c h . 5 3 , 40-43. D r i p p s , R . D . , C o m r o e , J . H . , 1947. T h e respiratory a n d c i r c u l a t o r y response o f n o r m a l m a n to i n h a l a t i o n o f 7.6 and 10.4 per cent C O v w i t h a c o m p a r i s o n o f the m a x i m a l v e n t i l a t i o n produced  by  severe  muscular  exercise,  inhalation  of  CO2  and  maximal  voluntary  h y p e r v e n t i l a t i o n . A m . J . P h y s i o l . 149, 4 3 - 5 1 . Dulla, C.G.,  D o b e l i s , P., P e a r s o n , T.,  Frenguelli, B.G.,  Staley, K . J . , M a s i n o , S . A .  2005.  A d e n o s i n e and A T P l i n k Pco2 to c o r t i c a l e x c i t a b i l i t y v i a p H . N e u r o n 4 8 , 1 0 1 1 - 1 0 2 3 E i s e l e , J . H . , E g e r , E . L , M u a l l e m , M . , 1967. N a r c o t i c properties o f c a r b o n d i o x i d e i n the d o g . Anesthesiology 28, 856-864. F a w e l l , J . K . , T h o m s o m , C . , C o o k e , L., 1972. R e s p i r a t o r y artefact p r o d u c e d b y c a r b o n d i o x i d e and pentobarbitone s o d i u m euthanasia i n rats. L a b . A n i m . 6, 3 2 1 - 3 2 6 . F e n g , Y . , S i m p s o n , T. L., 2 0 0 3 . N o c i c e p t i v e sensation and sensitivity e v o k e d f r o m h u m a n c o r n e a and c o n j u n c t i v a s t i m u l a t e d b y CO2. Invest. O p h t h . V i s . S c i . 4 4 , 5 2 9 - 5 3 2 . F o w l e r , W . S . , 1954. B r e a k i n g p o i n t o f b r e a t h - h o l d i n g . J . A p p l . P h y s i o l . 6, 5 3 9 - 5 4 5 . Fraser, D . , W e a r y , D . M . , P a j o r , E . A . , M i l l i g a n , B . N . , 1997. A s c i e n t i f i c c o n c e p t i o n o f a n i m a l w e l f a r e that reflects ethical concerns. A n i m . W e l f a r e 6, 1 8 7 - 2 0 5 . F u k u d a , Y . , Satao, A , S u z u k i , A , T r z e b s k i , A . , 1989. A u t o n o m i c nerve and c a r d i o v a s c u l a r response to c h a n g i n g b l o o d o x y g e n and c a r b o n d i o x i d e levels i n the rat. J . A u t o n . N e r v . Syst! 2 8 , 6 1 - 7 4 . . 32  Gerritzen,  M.A.,  L a m b o o i j , E.,  Hillebrand,  S.J.W.,  Lanhaar,  J.A.C.,  Pieterse, C ,  2000.  B e h a v i o r a l responses o f broilers to different gaseous atmospheres. P o u l t r y S c i . 7 9 , 9 2 8 - 9 3 3 . Gifford, R . G . , Monyer, H., Christine, C . W . , C h o i , D . W . a c t i v a t i o n , glutamate n e u r o t o x i c i t y ,  A c i d o s i s reduces N M D A  and o x y g e n - g l u c o s e  receptor  deprivation neuronal injury  in  c o r t i c a l cultures. B r a i n R e s . 5 0 6 , 3 3 9 - 3 4 2 . H a c k b a r t h , H . , K u p p e r s , N . , B o h n e t , W . , 2 0 0 0 . E u t h a n a s i a o f rats w i t h c a r b o n d i o x i d e — a n i m a l w e l f a r e aspects. L a b . A n i m . 3 4 , 9 1 - 9 6 . H a l d a n e , J . , S m i t h , F., 1892. J . P a t h o l . B a c t e r i o l . 168. C i t e d b y H i l l a n d F l a c k , 1 9 0 8 . H a u s e r , M . D . 1996. T h e E v o l u t i o n o f C o m m u n i c a t i o n . M I T Press, C a m b r i d g e , Massachusetts. Herman,  J.P.  &  Cullinan,  W.E.,  1997. N e u r o c i r c u i t r y  of  stress:  central  control  of  the  h y p o t h a l a m o - p i t u i t a r y - a d r e n o c o r t i c a l axis. T r e n d s N e u r o s c i . 2 0 , 7 8 - 8 4 . H e r m a n , J . P., P r e w i t t , C . M . F., C u l l i n a n , W . E., 1996. N e u r o n a l c i r c u i t r e g u l a t i o n o f the h y p o t h a l a m o - p i t u i t a r y - a d r e n o c o r t i c a l stress a x i s . C r i t . R e v . N e u r o b i o l . 10, 3 7 1 - 3 9 4 . H e w e t t , T . A . , K o v a c s , M . S . , A r t w o h l , J . E . , Bennett, B . T . ,  1 9 9 3 . A c o m p a r i s o n o f euthanasia  methods i n rats, u s i n g c a r b o n d i o x i d e i n p r e - f i l l e d a n d fixed flow rate f i l l e d c h a m b e r s . L a b . A n i m . Sci. 43, 579-582. H i l l , L., F l a c k , M . , 1908. T h e effect o f excess o f c a r b o n d i o x i d e and o f want o f o x y g e n u p o n the respiration and the c i r c u l a t i o n . J . P h y s i o l . 3 7 , 7 7 - 1 1 1 . H i r a t a , H . , H u , J . W . , Bereiter, D . A . ,  1 9 9 9 . R espon ses o f m e d u l l a r y d o r s a l h o r n neurons to  c o r n e a l s t i m u l a t i o n b y CO2 pulses i n the rat. J . N e u r o p h y s i o l . 8 2 , 2 0 9 2 - 2 1 0 7 . H o d g e s , M . R . , Forster, H . V . ,  Papanek, P.E., D w i n e l l , M . R . & H o g a n , G . E . , 2002. Ventilatory  phenotypes a m o n g four strains o f adult rats. J . A p p l . P h y s i o l . 9 3 , 9 7 4 - 9 8 3 . H o r n e t t , T . D . , H a y n e s , A . R . , 1984. C o m p a r i s o n o f c a r b o n dioxide/air m i x t u r e and nitrogen/air m i x t u r e for the euthanasia o f rodents. D e s i g n o f a system for i n h a l a t i o n euthanasia. A n i m a l Technology 35, 93-99. 33  H s u , K.,  Liang, Y., Huang, C ,  2 0 0 0 . I n f l u e n c e o f an extracellualr a c i d o s i s o n  synaptic t r a n s m i s s i o n a n d l o n g - t e r m potentiation i n the C A I  excitatory  r e g i o n o f rat h i p p o c a m p a l  slices. J . N e u r o s c i . R e s . 6 2 , 4 0 3 - 4 1 5 . Iwarsson, K., Rehbinder, C ,  1 9 9 3 . A study o f different euthanasia techniques i n g u i n e a p i g s ,  rats, and m i c e . A n i m a l response and p o s t m o r t e m f i n d i n g s . S c a n . J . L a b . A n i m . S c i . 2 0 , 1 9 1 205. J o u r d a n , D . , A r d i d , D . , C h a p u y , E., E s c h a l i e r , A . , L e B a r s , D . , 1 9 9 5 . A u d i b l e and ultrasonic v o c a l i z a t i o n e l i c i t e d b y single electrical n o c i c e p t i v e s t i m u l i to the t a i l i n the rat. P a i n 6 3 , 237-249. J o u r d a n , D . , A r d i d , D . , C h a p u y , E., L e B a r s , D . , E s c h a l i e r , A . , 1 9 9 8 . E f f e c t o f analgesics o n audible and ultrasonic p a i n - i n d u c e d v o c a l i z a t i o n i n the rat. L i f e S c i . 6 3 , 1 7 6 1 - 1 7 6 8 . Kaltwasser, M.T.,  1 9 9 0 . S t a r t l e - i n d u c i n g acoustic s t i m u l i e v o k e u l t r a s o n i c v o c a l i z a t i o n i n the  rat. P h y s i o l . B e h a v . 4 8 , 1 3 - 1 7 . K c , P., H a x h i u , M . A . , Trouth, C O . , Balan, K . V . , Anderson, W . A . , M a c k , S.O., 2002. C 0 2  i n d u c e d c - F o s e x p r e s s i o n i n h y p o t h a l a m i c v a s o p r e s s i n c o n t a i n i n g neurons. R e s p . P h y s i o l . 129,289-296. Kitchen, H., A r o n s o n , A . L . , Bittle, J.L., M c P h e r s o n , C . W . , M o r t o n , D . B . , Pakes, S.P., R o l l i n , B . E . , R o w a n , A . N . , Sechzer, J.A., Vanderlip, J.E., W i l l , J . A . , Clark, A . S . , G l o y d , J.S., 1987. P a n e l report o n the C o l l o q u i u m o n r e c o g n i t i o n a n d a l l e v i a t i o n o f a n i m a l p a i n and distress. J . A m . Vet. M e d . Assoc. 191, 1186-1191. K l u m p , G . M . , Shalter, M . D . , 1984. A c o u s t i c b e h a v i o u r o f b i r d s a n d m a m m a l s i n the predator context: I. F a c t o r s a f f e c t i n g the structure o f a l a r m signals. II. T h e f u n c t i o n a l s i g n i f i c a n c e and e v o l u t i o n o f a l a r m signals. Z . T i e r p s y c h o l . 6 6 , 1 8 9 - 2 2 6 . C i t e d i n H a u s e r , M . D . 1996 T h e E v o l u t i o n o f C o m m u n i c a t i o n , M I T Press, C a m b r i d g e , p p . 4 1 3 - 4 1 9 . K n u t s o n , B . , B u r g d o r f , J . , P a n k s e p p , J . , 2 0 0 2 . U l t r a s o n i c v o c a l i z a t i o n s as i n d i c e s o f affective 34  states i n rats. P s y c h o l . B u l l . 128, 9 6 1 - 9 7 7 . K o h l e r , I., M e i e r , R., B u s a t o , A . , N e i g e r - A e s c h b a c h e r , G . , S c h a t z m a n n , U . ,  1999. Is c a r b o n  d i o x i d e (CO2) a u s e f u l short a c t i n g anaesthetic for s m a l l laboratory a n i m a l s ? L a b . A n i m . 3 3 , 155-161. K o o l h a s , J . M . , K o r t e , S . M . , D e B o e r , S . F . , v a n der V e g t , B . J . , v a n R e e n e n , C . G . , H o p s t e r , H . , de J o n g , L C , R u i s , M . A . W . , B l o k h u i s , H . J . , 1999. C o p i n g styles i n a n i m a l s : current status i n b e h a v i o r a n d s t r e s s - p h y s i o l o g y . N e u r o s c i . B i o b e h a v . R. 2 3 , 9 2 5 - 9 3 5 . L e a c h , M . C , B o w e l l , V . A . , A l l a n , T . F . , M o r t o n , D . B . , 2 0 0 2 a . A v e r s i o n to gaseous euthanasia agents i n rats a n d m i c e . C o m p a r a t i v e M e d . 5 2 , 2 4 9 - 2 5 7 . L e a c h , M . C , B o w e l l , V . A . , A l l a n , T . F . , M o r t o n , D . B . , 2 0 0 2 b . Degrees o f a v e r s i o n s h o w n b y rats a n d m i c e to different concentrations o f i n h a l a t i o n a l anaesthetics. V e t . R e c . 1 5 0 , SOSSIS. L e e , J . , T a i r a , T., P i h l a j a , P., R a n s o m , B . R . , K a i l a , K . ,  1996. E f f e c t s o f CO2 o n  excitatory  t r a n s m i s s i o n apparently caused b y changes i n intracellular p H i n the rat h i p p o c a m p a l s l i c e . Brain Res. 706, 210-216. Liotti, M . , Brannan, S., Egan, G.,  Shade, R.,  M a d d e n , L.,  Abplanalp, B., Robillard,  R.,  L a n c a s t e r , J . , Z a m a r r i p a , F . E . , F o x , P.T., D e n t o n , D . , 2 0 0 1 . B r a i n responses associated w i t h c o n s c i o u s n e s s o f breathlessness (air hunger). P r o c . N a t . A c a d . S c i . 9 8 , 2 0 3 5 - 2 0 4 0 . L u s h , M . T . , J a n s o n - B j e r k l i e , S . , C a r r i e r i , V . K . , L o v e j o y , N . , 1 9 8 8 . D y s p n e a i n the ventilatorassisted patient. Heart L u n g , 17, 5 2 8 - 5 3 5 . M a r o t t a , S . F . , S i t h i c h o k e , N . , G a r c y , A . M . , Y u , M . , 1976. A d r e n o c o r t i c a l responses o f rats to acute  hypoxic  and  hypercapnic  stresses  after  treatment  with  aminergic  agents.  Neuroendocrinology 20, 182-192. M a r t o f t , L., L o m h o l t , L., K o l t h o f f , C , R o d r i g u e s , B . E . , Jensen, E . W . , Jorgensen, P . F . , P e d e r s e n , H . D . , F o r s l i d , A . , 2 0 0 2 . E f f e c t s o f CO2 anaesthesia o n central nervous system a c t i v i t y i n 35  swine. Lab. A n i m . 36, 115-126. M a r t o f t , L., S t o d k i l d e - J o r g e n s e n , H . , F o r s l i d , A . , P e d e r s e n , H . D . & Jorgensens, P . F . , 2 0 0 3 . CO2 i n d u c e d acute respiratory a c i d o s i s a n d intracellular p H : a  3 I  P N M R study i n s w i n e . L a b .  Anim. 37,241-248. M a s o n , G . J . , C o o p e r , J . , C l a r e b r o u g h , C , 2001 Frustrations o f f u r - f a r m e d m i n k . N a t u r e 4 1 0 , 3 5 36. M a s u d a , A . , O h y a b u , Y . , K o b a y a s h i , T., Y o s h i n o , C , S a k a k i b a r a , Y . , K o m a t s u , T., H o n d a , Y . , 2 0 0 1 . L a c k o f positive' interaction b e t w e e n CO2 and h y p o x i c s t i m u l a t i o n for Pco2 -  VAS  response slope i n h u m a n s . R e s p . P h y s i o l . 126, 1 7 3 - 1 8 1 . M e y e r , J . S . , G o t o h , F., T a z a k i , Y . , 1 9 6 1 . CO2 narcosis: a n e x p e r i m e n t a l study. N e u r o l o g y 1 1 , 524-537. M e y e r , J . S . , G o t o h , F., T o m i t a , M . , 1966. A c u t e respiratory a c a d e m i a . C o r r e l a t i o n o f j u g u l a r b l o o d c o m p o s i t i o n and e l e c t r o e n c e p h a l o g r a m d u r i n g CO2 narcosis. N e u r o l o g y 16, 4 6 3 - 4 7 4 . M o b e r g , G . P . , 2 0 0 0 . B i o l o g i c a l responses to stress: i m p l i c a t i o n s for a n i m a l w e l f a r e . In: M o b e r g , G . P . , M e n c h , J . A . (eds), B i o l o g y o f A n i m a l Stress, C A B I P u b l i s h i n g , N e w Y o r k , p p . 1-21 Montgomery,  K.C.,  1955. T h e  r e l a t i o n between  fear i n d u c e d  by  novel  stimulation  and  exploratory behavior. J. C o m p . Physiol. P s y c h o l . 4 8 , 254-260.  V M o o s a v i , S . H . , G o l e s t a n i a n , E., B i n k s , A . P . , L a n s i n g , R . W . , B r o w n , R., B a n z e t t , R . B . , 2 0 0 3 . H y p o x i c and h y p e r c a p n i c drives to breathe generate equivalent levels o f air hunger  in  humans. J. A p p l . Physiol. 94, 141-154. N a t i o n a l R e s e a r c h C o u n c i l , 2 0 0 3 . G u i d e l i n e s for the C a r e and U s e o f M a m m a l s i n N e u r o s c i e n c e and B e h a v i o r a l R e s e a r c h . T h e N a t i o n a l A c a d e m i e s Press, W a s h i n g t o n , D . C . , p. 16. N i e l s o n , M . , S m i t h , H . , 1 9 5 2 . Studies o n the r e g u l a t i o n o f respiration i n acute h y p o x i a . A c t a . Physiol. Scand. 24, 293-313. Pasternak, M . ,  Bountra,  C,  Voipio,  J., Kaila, 36  K.,  1992. Influence  o f extracellular  and  intracellular  pH  on  GABA-gated  chloride  conductance  in  crayfish  muscle  fibers.  Neuroscience 47, 921-929. Pecaut, M . J . , S m i t h , A . L . , Jones, T . A . , G r i d l e y , D . S . , 2 0 0 0 . M o d i f i c a t i o n o f i m m u n o l o g i c and h e m a t o l o g i c variables b y m e t h o d o f CO2 euthanasia. C o m p a r a t i v e M e d . 5 0 , 5 9 5 - 6 0 2 . P e p p e l , P., A n t o n , F., intranasal n o x i o u s  1 9 9 3 . R e s p o n s e s o f rat m e d u l l a r y d o r s a l h o r n neurons chemical  s t i m u l a t i o n : effects  of  stimulus  intensity,  following  duration,  and  interstimulus interval. J. N e u r o p h y s i o l . 70, 2260-2275. Raff, H., Roarty, T.P.,  1988. R e n i n , A C T H ,  and aldosterone d u r i n g acute h y p e r c a p n i a and  h y p o x i a i n c o n s c i o u s rats. A m . J . P h y s i o l . 2 5 4 , R 4 3 1 - R 4 3 5 . R a j , A . B . , 1996. A v e r s i v e reactions o f turkeys to a r g o n , c a r b o n d i o x i d e and a m i x t u r e o f c a r b o n d i o x i d e and argon. V e t . R e c . 1 3 8 , 5 9 2 - 5 9 3 . R a j , A . B . , Johnson, S.P., Wotton, S . B . , Mclnstry, J.L.,  1 9 9 7 . W e l f a r e i m p l i c a t i o n s o f gas  s t u n n in g p i g s : 3 . T h e t i m e to loss o f S o m a t o s e n s o r y e v o k e d potentials and spontaneous e l e c t r o c o r t i c o g r a m o f p i g s d u r i n g exposure to gases. V e t . J . 1 5 3 , 3 2 9 - 3 3 9 . Raj, A . B . M . ,  Wotton, S.B., Gregory, N.G.,  1992. Changes  i n the somatosensory  evoked  potential and spontaneous e l e c t r o e n c e p h a l o g r a m o f hens d u r i n g stunning w i t h a c a r b o n d i o x i d e a n d argon m i x t u r e . B r i t . V e t . J . 4 8 , 1 4 7 - 1 5 6 . R a j , A . B . , W o t t o n , S . B . , M c K i n s t r y , J . L . , H i l l e b r a n d , S . J . , Pieterse, C ,  1 9 9 8 . C h a n g e s i n the  somatosensory e v o k e d potentials and spontaneous e l e c t r o e n c e p h a l o g r a m o f b r o i l e r c h i c k e n s d u r i n g exposure to gas m i x t u r e s . B r i t . P o u l t r y S c i . 3 9 , 6 8 6 - 6 9 5 . Raj, A . B . M . ,  Gregory, N . G . ,  1995. W e l f a r e i m p l i c a t i o n s o f the gas stunning o f pigs  1.  D e t e r m i n a t i o n o f a v e r s i o n to the i n i t i a l i n h a l a t i o n o f c a r b o n d i o x i d e or argon. A n i m . W e l f a r e 4,273-280. R a j , M . , G r e g o r y , N . G . , 1994. A n e v a l u a t i o n o f h u m a n e gas stun n i n g methods for turkeys. V e t . Rec. 135, 222-223. 37  R o w a n , A . N . , Stephens, M L . , D o l i n s , F., G l e a s o n , A . , D o n l e y , L.,  1998. A n i m a l welfare  perspectives o n p a i n and distress management i n research and testing. In: P r o c e e d i n g s for P a i n M a n a g e m e n t and H u m a n e E n d p o i n t s , a w o r k s h o p o f T h e J o h n s H o p k i n s Center for A l t e r n a t i v e s to A n i m a l T e s t i n g , h e l d N o v e m b e r 2 - 3 , 1 9 9 8 , W a s h i n g t o n , D C . A v a i l a b l e at: http://altweb.ihsph.edu/meetings/pain/rowan.htm accessed M a y 2 0 0 6 . R u s h e n , J . , 1 9 9 6 . U s i n g a v e r s i o n l e a r n i n g techniques to assess the m e n t a l state, s u f f e r i n g and welfare o f farm animals. J. A n i m . S c i . 74, 1990-1995. S e l y e , H . 1 9 7 5 . C o n f u s i o n and c o n t r o v e r s y i n the stress f i e l d . J . H u m . Stress 1, 3 7 - 4 4 . S h a r p , J . , A z a r , T., L a w s o n , D . , 2 0 0 6 . C o m p a r i s o n o f c a r b o n d i o x i d e , a r g o n , and nitrogen for i n d u c i n g u n c o n s c i o u s n e s s . o r euthanasia o f rats. J . A m . A s s o c . L a b . A n i m . S c i . 4 5 , 2 1 - 2 5 . S h a r p , J . , Z a m m i t , T., A z a r , T., L a w s o n , D . , 2 0 0 2 . S t r e s s - l i k e responses to c o m m o n procedures i n rats h o u s e d alone or w i t h other rats. C o n t e m p . T o p . L a b . A n i m . 4 1 , 8 - 1 4 . S h a r p , J . , Z a m m i t , T., A z a r , T., L a w s o n , D . , 2 0 0 3 . S t r e s s - l i k e responses to c o m m o n procedures i n i n d i v i d u a l l y and g r o u p - h o u s e d f e m a l e rats. C o n t e m p . T o p . L a b . A n i m . 4 2 , 9 - 1 8 . S h e a , S . A . , H a t t y , H . R . , B a n z e t t , R . B . , 1996. S e l f - c o n t r o l o f l e v e l o f m e c h a n i c a l v e n t i l a t i o n to m i n i m i z e C O 2 i n d u c e d air hunger. R e s p . P h y s i o l . 1 1 3 - 1 2 5 . S m i t h , W . , H a r r a p , S . B . , 1997. B e h a v i o u r a l a n d c a r d i o v a s c u l a r responses o f rats to euthanasia u s i n g c a r b o n d i o x i d e gas. L a b . A n i m . 3 1 , 3 3 7 - 3 4 6 . S o m e r o , G . N . , 1 9 8 6 . P r o t o n s , o s m o l y t e s , and fitness o f internal m i l i e u for p r o t e i n f u n c t i o n . A m . J. Physiol. 2 5 1 . R 1 9 7 - R 2 1 3 . T a n g , C M . , D i c h t e r , M . , M o r a d , M . , 1 9 9 0 . M o d u l a t i o n o f the N - m e t h y l - D - a s p a r t a t e c h a n n e l b y extracellular H . P r o c . N a t l . A c a d . S c i . 8 7 , 6 4 4 5 - 6 4 4 9 . +  T e r l o u w , E . M . C , S c h o u t e n , G . P . , L a d e w i g , J . , 1997. P h y s i o l o g y . In: A p p l e b y , M . C ,  Hughes,  B . O . (eds), A n i m a l W e l f a r e , C A B I P u b l i s h i n g , W a l l i n g f o r d , p p . 1 4 3 - 1 5 8 . Thomas, D.A.,  Takahashi, L.K.,  Barfield, R.J., 38  1 9 8 3 . A n a l y s i s o f ultrasonic  vocalizations  emitted b y intruders d u r i n g aggressive encounters a m o n g rats (Rattus n o r v e g i c u s ) . J . C o m p . Psychol. 97,201-206. T h u r a u f , N . , G u n t h e r , M . , P a u l i , E., K o b a l , G . , 2 0 0 2 . S e n s i t i v i t y o f the negative m u c o s a l potential to the t r i g e m i n a l target s t i m u l u s CO2. B r a i n R e s . 9 4 2 , 2 7 - 8 6 . Thurauf, N . ,  H u m m e l , T.,  Kettenmann, B.,  Kobal,  G.,  1993. Nociceptive  and  reflexive  responses r e c o r d e d f r o m the h u m a n nasal m u c o s a . B r a i n R e s . 6 2 9 , 2 9 3 - 2 9 9 . Toates, F., 1 9 9 7 . Stress: C o n c e p t u a l and B i o l o g i c a l Perspectives. C h i c h e s t e r , J o h n W i l e y and Sons. T o m b a u g h , G . C . , S o m j e n , G . C . , 1996. E f f e c t s o f extracellular p H o n v o l t a g e - g a t e d N a , K , and +  Ca  +  United  +  currents i n isolated rat C A I neurons. J . P h y s i o l . 4 9 3 , 7 1 9 - 7 3 2 . Kingdom  Home  Office,  1995. T h e  Welfare  of  Animals  (Slaughter  or  Killing)  R e g u l a t i o n s 1 9 9 5 , H e r M a j e s t y ' s Stationery O f f i c e , N o r w i c h . U n i t e d K i n g d o m H o m e O f f i c e , 1997. T h e H u m a n e K i l l i n g o f A n i m a l under S c h e d u l e 1 to the A n i m a l s ( S c i e n t i f i c Procedures) A c t 1986 C o d e o f P r a c t i c e , H e r M a j e s t y ' s Stationery O f f i c e , Norwich. V e l i s e k , L., 1 9 9 8 . E x t r a c e l l u l a r a c i d o s i s and h i g h levels o f c a r b o n d i o x i d e suppress synaptic t r a n s m i s s i o n a n d prevent the i n d u c t i o n o f l o n g - t e r m potentiation i n the C A I  r e g i o n o f rat  h i p p o c a m p a l slices. H i p p o c a m p u s 8, 2 4 - 3 2 . V i n g e r h o e t s , J . J . M . , 1985. T h e role o f the parasympathetic d i v i s i o n o f the a u t o n o m i c nervous s y s t e m i n stress and e m o t i o n s . Int. J . P s y c h o s o m a t i c s 3 2 , 2 8 - 3 3 . Vivian,  J.A.,  Miczek,  K.A.,  1991. Ultrasounds  during  morphine  withdrawal  in  rats.  P s y c h o p h a r m a c o l o g y 104, 1 8 7 - 1 9 3 . W a l l a c e , K . J . , R o s e n , J . B . , 2 0 0 0 . Predator o d o r as an u n c o n d i t i o n e d fear s t i m u l u s i n rats: e l i c i t a t i o n o f f r e e z i n g b y t r i m e t h y l t h i a z o l i n e , a c o m p o n e n t o f f o x feces. B e h a v . N e u r o s c i . 114,912-922. 39  Webster, A . B . , Fletcher, D.L.,  2 0 0 4 . A s s e s s m e n t o f the a v e r s i o n o f hens to different gas  atmospheres u s i n g a n a p p r o a c h - a v o i d a n c e test. A p p l . A n i m . B e h a v . S c i . 8 8 , 2 7 5 - 2 8 7 . W i d d i c o m b e , J . G . , 1986 R e f l e x e s f r o m the upper respiratory tract. In: H a n d b o o k o f P h y s i o l o g y , The Respiratory System, N . S . Cherniak, J . G . W i d d i c o m b e (Eds.), A m e r i c a n Physiological Socienty, Bethesda, 363-394. W o o d b u r y , D . M . , R o l l i n s , L.T., Tanner, G . S . , Brodie, D . A . ,  Gardner, M . D . , H i r s c h i , W . L . , H o g a n , J.R., Rallison,  M.L.,  1958. Effects o f carbon dioxide on brain excitability  and  electrolytes. A m . J . P h y s i o l . 1 9 2 , 7 9 - 9 0 . Yavari,  P., M c C u l l o c h , P . F . ,  Panneton, W . M . ,  1996. T r i g e m i n a l l y - m e d i a t e d  alteration  of  cardiorespiratory r h y t h m s d u r i n g nasal a p p l i c a t i o n o f c a r b o n d i o x i d e i n the rat. J . A u t o n . N e r v . Syst. 6 1 , 1 9 5 - 2 0 0 . Youngentob,  S.L., Hornung,  D.E.,  M o z e l l , M . M . , 1991. Determination o f carbon  detection thresholds i n trained rats. P h y s i o l . . B e h a v . 4 9 , 2 1 - 2 6 .  40  dioxide  CHAPTER 2: Behavioural responses of rats to gradual-fill carbon dioxide euthanasia and reduced oxygen concentrations 2.1 Introduction M e t h o d s c o m m o n l y used for euthanasia o f s m a l l laboratory rodents i n c l u d e p h y s i c a l S  techniques, injectable anaesthetics, and exposure to anaesthetic and non-anaesthetic gases. Exposure  to CO2  (ANZCCART,  is one o f the m o s t w i d e l y  r e c o m m e n d e d euthanasia methods for  rats  1 9 9 3 ; A V M A , 2 0 0 0 ; C a n a d i a n C o u n c i l o n A n i m a l C a r e , 1 9 9 3 ; C l o s e et a l . ,  1 9 9 7 ; U K H o m e O f f i c e , 1997). R a t s are either p l a c e d into a c h a m b e r p r e - f i l l e d w i t h gas or the gas is g r a d u a l l y i n t r o d u c e d into a n a i r - f i l l e d c h a m b e r , a n d this results i n narcosis due to the properties o f CO2 f o l l o w e d b y death. B o t h methods are r e l a t i v e l y easy to p e r f o r m , i n e x p e n s i v e , safe for laboratory w o r k e r s , and i n v o l v e little h a n d l i n g and restraint o f a n i m a l s . I d e a l l y , a euthanasia m e t h o d s h o u l d also result i n a q u i c k death w i t h m i n i m a l p a i n and distress before loss o f c o n s c i o u s n e s s , but it is not clear whether CO2 euthanasia meets these last criteria. CO2 f o r m s c a r b o n i c a c i d w h e n it c o m e s into contact w i t h m o i s t u r e . It begins to stimulate n o c i c e p t o r s i n rat nasal m u c o s a at CO2 concentrations above 2 5 % , a n d the threshold for the m a j o r i t y o f n o c i c e p t o r s is between 3 7 % and 5 0 % CO2 ( A n t o n et a l . , 1 9 9 1 ; P e p p e l & A n t o n , 1993). In h u m a n s , CO2 is detectable and begins to b e c o m e p a i n f u l at the c o r n e a , c o n j u n c t i v a and the nasal m u c o s a at concentrations b e t w e e n 30%) and 5 4 % ( A n t o n et a l . , 1 9 9 2 ; C h e n et a l , 1 9 9 5 ; F e n g & S i m p s o n , 2 0 0 3 ) . H i g h CO2 concentrations c a n also cause d y s p n e a , or shortness  of  breath, w h i c h i n c l u d e s the sensations o f b o t h air hunger and increased b r e a t h i n g effort ( L a n s i n g et a l . , 2 0 0 0 ) . A t l o w levels o f CO2 d y s p n e a c a n be o v e r c o m e b y ventilatory adjustments (Shea et a l , 1996), but spontaneously b r e a t h i n g h u m a n s report this sensation at CO2 concentrations o f o n l y 8%> ( D r i p p s & C o m r o e , 1 9 4 7 ; L i o t t o et a l . , 2 0 0 1 ) and severity increases w i t h i n c r e a s i n g A version of this chapter has been published. Niel, L . , Weary, D . M . , 2006. Behavioural responses of rats to gradual-fill carbon dioxide euthanasia and reduced oxygen concentrations. Appl. Anim. Behav. Sci. (in press). 2  41  CO2 concentrations (Banzett et a l . , 1996). A d d i t i o n o f CO2 to a c h a m b e r causes a r e d u c t i o n i n O2 l e v e l s w i t h d i s p l a c e m e n t o f air, w h i c h m a y also cause d y s p n e a . T h e O2 concentration i n a m b i e n t air i s 2 0 . 9 % ,  a n d h u m a n s report d y s p n e a at O2 l e v e l s o f less than 8 %  when  c o m p e n s a t o r y b r e a t h i n g is constrained ( M o o s a v i et a l . , 2 0 0 3 ) . T h i s sensation w a s a l l e v i a t e d w i t h spontaneous breathing, but O2 concentrations less than 7 % were not e x a m i n e d . D u r i n g p r e f i l l CO2 euthanasia rats are e x p o s e d to CO2 concentrations above 7 0 % , so it seems l i k e l y that rats experience b o t h p a i n and d y s p n e a u s i n g this m e t h o d . H o w e v e r , d u r i n g g r a d u a l - f i l l CO2 euthanasia rats t y p i c a l l y lose consciousness at CO2 concentrations b e l o w 4 0 % ( S m i t h & H a r r a p , 1997) a n d so m a y a v o i d some o f these negative sensations. If CO2 does cause p a i n or d y s p n e a i n rats, 'distress' b e h a v i o u r s w o u l d be expected d u r i n g exposure. T h e s e c o u l d i n c l u d e b e h a v i o u r s associated w i t h p a i n s u c h as h e a d - s h a k i n g and r u b b i n g the nose and eyes, b e h a v i o u r s  associated w i t h gas a v o i d a n c e  s u c h as increased  e x p l o r a t i o n and escape attempts, and general distress b e h a v i o u r s s u c h as increases i n particular v o c a l i z a t i o n s . B e h a v i o u r a l studies o n rats d u r i n g CO2 euthanasia have generally  examined  responses to different methods o f CO2 d e l i v e r y , w i t h o u t c o m p a r i s o n to a c o n t r o l session. In these studies, some authors have reported a l a c k o f distress b e h a v i o u r s d u r i n g p r e - f i l l ( S m i t h a n d H a r r a p , 1997) a n d g r a d u a l - f i l l C 0  2  exposure ( H a c k b a r t h et a l . , 2 0 0 0 ; H o r n e t t &  Haynes,  1 9 8 4 ; S m i t h & H a r r a p , 1997). O t h e r studies have reported ' a g i t a t i o n and a s p h y x i a t i o n ' d u r i n g b o t h p r e - f i l l and g r a d u a l - f i l l exposure ( C o e n e n et a l . , 1995), a n d ' m i l d to moderate stress' d u r i n g p r e - f i l l exposure ( I w a r s s o n & R e h b i n d e r , 1993). H o w e v e r , these authors p r o v i d e  few  d e t a i l e d b e h a v i o u r a l d e s c r i p t i o n s or data to support their c o n c l u s i o n s . T h r e e studies h a v e t a k e n objective b e h a v i o u r a l measures d u r i n g b o t h CO2 and air exposure. B l a c k s h a w et a l . (1988) f o u n d that p r e - f i l l CO2 exposure caused a decrease i n activity. L e a c h et a l . (2002) e x a m i n e d b e h a v i o u r a l responses a n d a v e r s i o n o f rats to static CO2 concentrations greater than 2 5 . 5 % , and f o u n d that rats a v o i d e d CO2 exposure, and that it caused increased f a c e - w a s h i n g . B r i t t (1987) 42  e x a m i n e d responses to g r a d u a l - f i l l exposure and f o u n d that changes i n a c t i v i t y and w a l l c l i m b i n g v a r i e d w i t h strain, but that s h a k i n g a l w a y s increased. If d y s p n e a does o c c u r d u r i n g CO2 euthanasia, s o m e o f this effect m a y be due to r e d u c e d O2 l e v e l s . N o studies to date have m e a s u r e d the responses d f rats to O2 r e d u c t i o n at the levels that o c c u r d u r i n g g r a d u a l - f i l l euthanasia. H o w e v e r , studies u s i n g subjective assessments o f distress have c l a i m e d that O2 s u p p l e m e n t a t i o n decreases responses o f rats d u r i n g  pre-fill  ( I w a r s s o n & R e h b i n d e r , 1993) and g r a d u a l - f i l l ( C o e n e n et a l . , 1995) CO2 exposure. T h e a i m s o f the current study w e r e to determine whether rats s h o w b e h a v i o u r a l signs o f distress d u r i n g g r a d u a l - f i l l CO2 euthanasia and d u r i n g an equivalent r e d u c t i o n i n O2 l e v e l caused b y d i s p l a c i n g air w i t h argon. W e p r e d i c t e d that distress d u r i n g exposure w o u l d be a c c o m p a n i e d b y increased e x p l o r a t i o n , escape attempts and v o c a l i z a t i o n , and that p a i n at the m u c o s a l m e m b r a n e s and c o r n e a w o u l d be a c c o m p a n i e d b y head s h a k i n g a n d face w a s h i n g .  2.2 Materials and Methods 2.2.1 Subjects S i x t e e n 4 0 0 - 5 0 0 g, mature, m a l e Sprague D a w l e y rats were obtained as surplus stock (i.e. a n i m a l s already slated for euthanasia) f r o m the U B C R o d e n t B r e e d i n g U n i t . A n i m a l s were g r o u p - h o u s e d at 21°C under a 1 2 : 1 2 - h r l i g h t - d a r k c y c l e , and g i v e n ad l i b i t u m access to f o o d ( L a b D i e t 5 0 0 1 , P M I N u t r i t i o n International, Indiana, U S A )  and tap water. A l l testing w a s  c o n d u c t e d d u r i n g the l i g h t p o r t i o n o f the l i g h t - d a r k c y c l e .  2.2.2 Experimental Apparatus T h e euthanasia c h a m b e r w a s a 2 0 L p o l y p r o p y l e n e cage 2 0 . 5 c m h i g h , 4 5 . 5 c m l o n g and 24 c m w i d e at the top ( L a b P r o d u c t s Inc.), fitted w i t h a P l e x i g l a s l i d . T h e l i d had a gas inlet  43  centered at one e n d , t w o a i r outlets p o s i t i o n e d at the opposite e n d , and a gas s a m p l i n g tube inserted at the center o f the c h a m b e r to a depth o f h a l f the c h a m b e r height. T h e air outlets were c o v e r e d w i t h m e s h to prevent the rats f r o m p u s h i n g their noses outside the ch am ber. T h e b a c k a n d sides o f the c h a m b e r were c o v e r e d w i t h b l a c k paper so that the a n i m a l s c o u l d not see the p e r s o n c o n d u c t i n g the experiment. A r g o n , an inert gas, w a s used to d i s p l a c e air i n the C ^ - r e d u c t i o n treatment group. C a r b o n d i o x i d e a n d argon w e r e d e l i v e r e d to the c h a m b e r f r o m c o m p r e s s e d gas c y l i n d e r s  (Praxair,  R i c h m o n d , B . C . ) , w h i l e r o o m air w a s d e l i v e r e d v i a a n a i r c o m p r e s s o r situated i n a n a d j o i n i n g r o o m . T h e treatment gases were passed through a copper c o i l i n a r o o m temperature water bath to regulate the temperature o f the gas before it entered the ch am ber. P r e l i m i n a r y tests i n d i c a t e d that the c h a m b e r temperature d i d n o t drop d u r i n g the f i l l i n g process. F l o w rates o f the gases were measured b y a v a r i a b l e area f l o w m e t e r ( M o d e l V S B - 6 6 - B V , D w y e r Instruments, I n c . , M i c h i g a n ) , a n d m e a s u r e d f l o w rates f o r CO2 a n d argon w e r e adjusted f o r density b y the c o r r e c t i o n factors 0 . 8 1 2 a n d 0 . 8 5 2 respectively.  G a s concentrations i n the c h a m b e r  were  m o n i t o r e d d u r i n g the e x p e r i m e n t v i a a gas s a m p l i n g tube u s i n g a M o c o n L F 7 0 0 D O2 analyzer. It w a s a s s u m e d that a n y decrease i n O2 w a s directly  related to a decrease i n a i r a n d a  c o r r e s p o n d i n g increase i n the treatment gas. T h e r e f o r e the f o l l o w i n g f o r m u l a w a s u s e d t o calculate the c o n c e n t r a t i o n o f CO2 at s p e c i f i c t i m e p o i n t s (t = x ) d u r i n g the f i l l i n g process: [ C 0 ( , = *>] = 1 0 0 - ( 1 0 0 * ( [ 0 ( t = ) ] / [ 0 ( , = „)])). 2  2  X  2  2.2.3 A p p a r a t u s T e s t i n g B e f o r e starting the a n i m a l e x p e r i m e n t s , gas concentrations were m e a s u r e d i n different areas o f the e m p t y c h a m b e r d u r i n g the CO2 filling process. T h e c h a m b e r w a s d i v i d e d into t w e l v e sectors b y p a r t i t i o n i n g the c h a m b e r into three segments i n the x - p l a n e (length), t w o segments i n the y - p l a n e ( w i d t h ) a n d t w o segments i n the z - p l a n e (height). T h e gas s a m p l i n g tube 44  w a s p l a c e d i n the center o f the sector to be tested, a n d CO2 w a s added at a rate o f 3.5 L / m i n . T h e O2 concentration w a s r e c o r d e d e v e r y 5 s for 10 m i n . E a c h sector w a s tested three t i m e s . T h e sectors were tested i n r a n d o m order to account for m i n o r fluctuations i n e n v i r o n m e n t a l parameters.  2.2.4 E x p e r i m e n t a l P r o c e d u r e A n i m a l s were r a n d o m l y allocated to the CO2 or O2 r e d u c t i o n ( u s i n g argon) treatment groups (n = 8 for both). F o r b o t h groups, a n i m a l s w e r e first tested w i t h air exposure a n d then w i t h the treatment gas o n the f o l l o w i n g day. O n b o t h testing d a y s , e a c h a n i m a l w a s i n d i v i d u a l l y p l a c e d into the euthanasia-chamber for a 1 5 - m i n p e r i o d o f a c c l i m a t i z a t i o n d u r i n g w h i c h air w a s added to the c h a m b e r at a rate o f 3.5 L / m i n . T h e length o f the a c c l i m a t i z a t i o n p e r i o d w a s based o n p r e l i m i n a r y observations s h o w i n g h o w l o n g it t o o k a n i m a l s to cease e x p l o r a t i o n a n d b e c o m e i n a c t i v e after entry into the testing apparatus. A f t e r a c c l i m a t i z a t i o n , air f l o w ceased and either air, CO2 or argon f l o w w a s started at a rate o f 3.5 L / m i n . T h i s rate c o r r e s p o n d e d to 1 7 . 2 5 % o f the c h a m b e r v o l u m e b e i n g added per m i n u t e . A l t h o u g h air f l o w ceased d u r i n g treatment gas e x p o s u r e , the air c o m p r e s s o r r e m a i n e d o n throughout the e x p e r i m e n t i n order to c o n t r o l for noise effects. C02-treated a n i m a l s r e m a i n e d i n the c h a m b e r a n d w e r e m o n i t o r e d u n t i l death but argon-treated a n i m a l s w e r e r e m o v e d f r o m the c h a m b e r at the end o f the 1 0 5 - s o b s e r v a t i o n period.  P r e l i m i n a r y observations  s h o w e d that C02-treated  a n i m a l s ceased a l l  purposeful  m o v e m e n t w i t h i n this p e r i o d , so any relevant effects o f O2 d e p r i v a t i o n w o u l d be present d u r i n g this t i m e . T h e l e v e l o f O2 r e d u c t i o n resulting f r o m argon a d d i t i o n w a s not sufficient to cause u n c o n s c i o u s n e s s or death, and w a s used o n l y to simulate reductions i n O2 levels that o c c u r w h e n a c h a m b e r is filled w i t h CO2.  45  2.2.5 B e h a v i o u r a l A n a l y s i s The  euthanasia c h a m b e r  and  O2 meter  readout  were  video  recorded  during  the  e x p e r i m e n t a l procedure. E a c h a n i m a l w a s scored c o n t i n u o u s l y d u r i n g the last 105 s o f the a c c l i m a t i z a t i o n p e r i o d (baseline) a n d the first 105 s after gas flow b e g a n (exposure) for p r e d e f i n e d b e h a v i o u r s thought to relate to p a i n and distress ( T a b l e 2.1). T h e t i m e u n t i l c o m p l e t e r e c u m b e n c y a n d cessation o f breathing w a s also recorded. R e c u m b e n c y w a s d e f i n e d as a loss o f posture and m u s c l e tone. T h e t i m e u n t i l onset o f ataxia w a s not r e c o r d e d as it c o u l d not be accurately assessed i n a l l a n i m a l s . S o u n d data were c o l l e c t e d w i t h a ^ " c o n d e n s e r m i c r o p h o n e ( B r u e l and K j a e r , T y p e 4 1 3 5 ) , c o n n e c t e d to a p r e a m p l i f i e r ( B r u e l a n d K j a e r , T y p e 2 6 1 9 ) and a m e a s u r i n g a m p l i f i e r ( B r u e l and K j a e r , T y p e 2 6 3 6 ) . T h e s i g n a l w a s r e c o r d e d d i r e c t l y to a h i g h - c a p a c i t y hard d i s k at a rate o f 2 5 0 k H z u s i n g a 3 3 0 k H z P C I - D A S 1 2 0 0 / J R data a c q u i s i t i o n c a r d ( C o m p u t e r b o a r d s , and C B D i s k  1.4 software ( E n g i n e e r i n g  Design, Belmont, M A ) .  The  Inc.)  m i c r o p h o n e end w a s  suspended 0.5 c m into the euthanasia c h a m b e r t h r o u g h one o f the air outlets. S o u n d w a s r e c o r d e d d u r i n g the last 105 s o f the a c c l i m a t i z a t i o n p e r i o d (baseline) and the first 105 s after gas flow began (exposure). S o u n d a n a l y s i s w a s w i t h S I G N A L 4 . 0 ( E n g i n e e r i n g D e s i g n , B e l m o n t , M A ) . C a l l s were i d e n t i f i e d b y their f o r m and as b e i n g distinct f r o m ambient n o i s e . S o u n d s o f less than 5 m s duration were d i f f i c u l t to d i s t i n g u i s h f r o m a m b i e n t noise and were d i s c a r d e d f r o m the analysis. Suspected v o c a l i z a t i o n s were p l a y e d b a c k i n a frequency range a u d i b l e to h u m a n s b y s l o w i n g the r e c o r d i n g s b y a factor o f 0.05 to 0 . 1 . W h i s t l e - l i k e sounds  were  accepted as v o c a l i z a t i o n s  while  c l i c k s and other  m e c h a n i c a l sounds  were  d i s c a r d e d . R a t s produce w h i s t l e - l i k e U S V s by p u s h i n g air t h r o u g h a 1 to 2 m m h o l e f o r m e d v i a the tight o p p o s i t i o n o f the t w o v o c a l cords (Sanders et a l . , 2 0 0 1 ) . It is p o s s i b l e that rats use another p r o d u c t i o n m e c h a n i s m to produce c l i c k s , but i n this study it w a s not p o s s i b l e to adequately  distinguish  vocal  clicks  from  those 46  resulting  from  movement  in  the  cage.  V o c a l i z a t i o n s were  subjected to spectrographic a n a l y s i s to determine c a l l d u r a t i o n , peak  f r e q u e n c y , m a x i m u m f r e q u e n c y and m i n i m u m frequency.  2.2.6 Statistical Analysis The  b e h a v i o u r a l data were n o n - n o r m a l w i t h u n e q u a l v a r i a n c e s a n d c o u l d not be  corrected t h r o u g h the use o f transformations, so n o n - p a r a m e t r i c statistics were used f o r a n a l y s i s . H e a d - s h a k i n g w a s n o t o b s e r v e d i n a n y a n i m a l s a n d f a c e - w a s h i n g w a s o b s e r v e d o n l y i n t w o rats d u r i n g b a s e l i n e a n d o n e rat d u r i n g CO2 exposure, so these b e h a v i o u r s w e r e n o t i n c l u d e d i n the a n a l y s i s . CO2 a n d r e d u c e d 0  2  exposures were not c o n d u c t e d c o n c u r r e n t l y , therefore direct  .comparisons b e t w e e n the treatments were not p e r f o r m e d . T h e W i l c o x o n S i g n e d R a n k s Test w a s u s e d to c o m p a r e the change i n b e h a v i o u r f r o m baseline d u r i n g a i r exposure w i t h the change f r o m baseline d u r i n g either CO2 or reduced O2 exposure. A l l b e h a v i o u r a l data are presented as medians  with  2 5 % a n d 7 5 % interquartile  ranges.  V o c a l i z a t i o n parameters a n d t i m e to  r e c u m b e n c y and death are presented as means ± standard d e v i a t i o n s .  2.3 Results CO2 concentrations i n the e m p t y c h a m b e r rose a s y m p t o t i c a l l y d u r i n g the f i l l i n g process, r e a c h i n g 8 7 % after 6 0 0 s ( F i g . 2.1). C o n c e n t r a t i o n s o f CO2 tended to be greater at the b o t t o m o f the c h a m b e r t h a n the t o p , w i t h a peak d i f f e r e n t i a l o f 7%> (bottom at 9 . 4 % , t o p at 2 % ) after 2 0 s. A f t e r this t i m e , the concentrations began to converge. T h e r e were n o concentration differences i n the y - p l a n e d u r i n g f i l l i n g , a n d the x - p l a n e CO2 concentration d i f f e r e n t i a l b e t w e e n the p o s i t i o n s closest a n d furthest f r o m the gas inlet w a s less than 2 % throughout the filling process. D u r i n g the actual e x p e r i m e n t gas samples were t a k e n at a depth o f h a l f the c h a m b e r height, a n d  47  CO2 a n d argon concentrations consistently f e l l w i t h i n the range o f v a l u e s f o u n d  during  p r e l i m i n a r y testing. D u r i n g the baseline p e r i o d rats were generally i n a c t i v e , but a l l b e h a v i o u r a l measures increased after CO2 f l o w began ( F i g . 2.2). R e a r i n g and t o u c h i n g o f the nose to the l i d started to increase w i t h i n the first 15 s. A c t i v i t y and r e a r i n g p e a k e d b e t w e e n 15 s and 6 0 s, and t o u c h i n g o f the nose to the l i d , escape behaviours and v o c a l i z a t i o n s p e a k e d b e t w e e n 6 0 s and 9 0 s after CO2 began. O2 a n d CO2 concentrations reached a p p r o x i m a t e l y 2 0 % and 5 % after 15 s, 1 7 % and 20%) after 6 0 s, and 15%> and 28%) after 9 0 s. C o m p l e t e r e c u m b e n c y o c c u r r e d o n average 106 ± 12 s after f l o w i n i t i a t i o n , at O2 and CO2 concentrations o f a p p r o x i m a t e l y 14%> and 3 3 % . R a t s d i d not stop breathing u n t i l 4 4 3 ±  14 s into the procedure, at 0  2  and CO2 concentrations  of  a p p r o x i m a t e l y 4 % and 8 0 % . W h e n c o m p a r e d to air exposure, rats e x p o s e d to CO2 were m o r e a c t i v e , and s h o w e d s i g n i f i c a n t increases i n the f r e q u e n c y o f r e a r i n g , escape b e h a v i o u r s , v o c a l i z a t i o n s and i n the time  spent w i t h the nose c o n t a c t i n g the c h a m b e r l i d ( T a b l e 2.2). H o w e v e r , there  considerable v a r i a t i o n i n b e h a v i o u r a l response a m o n g a n i m a l s , as reflected b y the  was large  interquartile ranges f o r several b e h a v i o u r s . F o r e x a m p l e , d u r i n g the CO2 exposure p e r i o d one rat p e r f o r m e d 3 4 escape b e h a v i o u r s , w h i l e t w o others p e r f o r m e d none. V o c a l i z a t i o n s d u r i n g CO2 exposure consisted o f pure tones a n d c a l l s w i t h frequency m o d u l a t i o n , and these d i d not f a l l into o b v i o u s categories. C a l l s v a r i e d i n length and f r e q u e n c y , r a n g i n g f r o m 5 to 150 m s and 8.6 to 102.1 k H z . O n average the c a l l d u r a t i o n w a s 33 ± 28 m s , the average f r e q u e n c y range w a s 2 2 ± 19 k H z a n d the peak f r e q u e n c y w a s 4 4 ± 2 0 k H z . D u e to the s m a l l n u m b e r o f v o c a l i z a t i o n s p r o d u c e d d u r i n g air a n d argon e x p o s u r e , c a l l parameters were not c o m p a r e d b e t w e e n treatments. R a t s that were e x p o s e d to reduced O2 concentrations u s i n g argon e x h i b i t e d o n l y a s m a l l increase i n the t i m e spent w i t h the nose c o n t a c t i n g the c h a m b e r l i d and n o increases i n any other 48  v a r i a b l e s i n c o m p a r i s o n w i t h air exposure ( T a b l e 2.3). R a t s d i d not s h o w any signs o f a t a x i a or r e c u m b e n c y d u r i n g the 1 0 5 - s observational p e r i o d for this treatment.  2.4 Discussion In contrast to several p r e v i o u s studies o n g r a d u a l - f i l l CO2 euthanasia ( H a c k b a r t h et a l . , 2 0 0 0 ; H o r n e t t & H a y n e s , 1 9 8 4 ; S m i t h & H a r r a p , 1997), w e f o u n d that this procedure does cause b e h a v i o u r a l signs o f distress i n rats. N o t o n l y d i d the rats e x h i b i t general signs o f e x p l o r a t i o n s u c h as increased l o c o m o t i o n , r e a r i n g , and t o u c h i n g the nose to the l i d , they also s h o w e d escape b e h a v i o u r s and v o c a l i z a t i o n s . T h i s b e h a v i o u r a l response b e g a n w i t h i n the first 15 s after the start o f gas f l o w , demonstrating that rats respond to e v e n l o w (approx. 5 % ) concentrations o f CO2. W e d i d not observe increases i n h e a d - s h a k i n g or f a c e - w a s h i n g d u r i n g exposure, suggesting that a n i m a l s d i d not experience p a i n d u r i n g the t i m e w h e n they w e r e capable o f m o u n t i n g a b e h a v i o u r a l response. H o w e v e r , it is also p o s s i b l e that the m e a s u r e d behaviours were  not  appropriate i n d i c a t o r s o f upper respiratory p a i n i n rats. O u r results are consistent w i t h the subjective assessments o f ' a g i t a t i o n ' d u r i n g g r a d u a l f i l l reported b y C o e n e n et a l . ( 1 9 9 5 ) , and w i t h the increase i n a c t i v i t y reported b y B r i t t (1987) for Sprague D a w l e y rats. Interestingly, B r i t t reported a decrease i n a c t i v i t y for L i s t e r H o o d e d rats i n the same study, i n d i c a t i n g that strain m a y be an important factor i n response differences. O t h e r studies have reported f e w b e h a v i o u r a l changes i n W i s t a r ( B l a c k s h a w et a l . , 1 9 8 8 ; H o r n e t t & H a y n e s , 1984) and F - 3 4 4 rats ( H a c k b a r t h et a l . , 2 0 0 0 ) d u r i n g CO2 exposure. In our study, w e f o u n d c o n s i d e r a b l e v a r i a t i o n i n response a m o n g i n d i v i d u a l s , w i t h s o m e a n i m a l s d i s p l a y i n g n u m e r o u s escape attempts and others s h o w i n g little response d u r i n g the procedure. It is u n c l e a r whether this v a r i a t i o n indicates a difference i n the l e v e l o f distress r e s u l t i n g f r o m the p r o c e d u r e ,  49  or a d i f f e r e n c e i n h o w a n i m a l s r e s p o n d to distress. A l a c k o f b e h a v i o u r a l response does not necessarily indicate that the rats perceive the procedure as i n n o c u o u s . While  Leach  et  a l . (2002)  have  demonstrated  that  rats  will  avoid  static  CO2  concentrations o f 2 5 . 5 % and greater, this is the first study to s h o w increased escape attempts b y rats  during  CO2  euthanasia. T h e  design o f previous  euthanasia experiments  may  have  d i s c o u r a g e d e x p r e s s i o n o f escape b e h a v i o u r s . T h e rats i n our study h a d access to the c h a m b e r l i d and t i m e to e x p l o r e it t h o r o u g h l y before CO2 exposure. R a t s i n other studies m a y not have h a d access to the c h a m b e r l i d and i n m o s t other experiments rats w e r e u n f a m i l i a r w i t h the c h a m b e r at the t i m e o f exposure, w h i c h m a y have i n h i b i t e d escape attempts. O n l y one other study has attempted to measure v o c a l i z a t i o n s d u r i n g CO2 exposure, and n o c a l l s were detected (Britt, 1987). T h e author d i d not p r o v i d e details o n the s o u n d c o l l e c t i o n apparatus, so the s e n s i t i v i t y and f r e q u e n c y range o f the e q u i p m e n t is u n k n o w n . W e f o u n d that v o c a l i z a t i o n s were present at l o w levels d u r i n g baseline and argon exposure, but increased d u r i n g CO2 exposure. T h e m a j o r i t y o f studies o n rat U S V s have f o c u s e d o n what have been d e s c r i b e d as the 2 2 k H z (ranging f r o m a p p r o x i m a t e l y 2 0 - 3 0 k H z and 3 0 0 - 3 0 0 0 m s ) and 50 k H z (ranging f r o m a p p r o x i m a t e l y 3 0 - 7 0 k H z and < 80 m s ) c a l l s ( r e v i e w e d i n K n u t s o n et a l . , 2 0 0 2 ) . C a l l s i n the current study appear to be consistent w i t h those that have been d e s c r i b e d as 50 k H z c a l l s , and n o 22 k H z c a l l s were o b s e r v e d . C a l l s that have been g r o u p e d under the 50 k H z l a b e l i n p r e v i o u s studies have a c t u a l l y v a r i e d c o n s i d e r a b l y i n l e n g t h , frequency and shape, and f e w studies have p r o v i d e d detailed c a l l descriptions. T h i s m a k e s it d i f f i c u l t to determine whether these c a l l s are consistent across studies, a n d whether they are i n d i c a t i v e o f s i m i l a r states. A l t h o u g h the 50 k H z c a l l s have b e e n observed d u r i n g p o s i t i v e , contexts  s u c h as  a n t i c i p a t i o n o f r e w a r d ( B u r g d o r f et a l . , 2 0 0 0 ; K n u t s o n et a l . , 1 9 9 8 ; K n u t s o n et a l . , 1999), they have also been o b s e r v e d d u r i n g intermale aggression (Sales, 1 9 7 2 ; T h o m a s et a l . , 1983) and exposure to anesthetized c o n s p e c i f i c s ( B l a n c h a r d et a l . , 1993). These 50  occurrences  during  potentially negative contexts suggest that the calls may also be associated with distress. However, the CC^-exposed animals were more active and spent more time near the lid in the vicinity o f the microphone, and this may have improved our ability to detect calls during this condition. The increase in calls could also have been a by-product of increases in breathing frequency and depth that occur during hypercapnia, but the effects of breathing changes on U S V production has not previously been examined. The densities of the gases used in the current study were higher than air and may also have affected U S V characteristics. Roberts (1975) found that a reduction in gas density results in an increase in fundamental frequency and a decrease in amplitude o f U S V s , and it is therefore likely that an increase in gas density would have the opposite effect. However, this effect is due to changes in the speed of sound in gases with different densities, and the effect of helium is much greater than the effects of either CO2 or argOn. The speed o f sound in air, CO2 and argon at 2 1 ° C is approximately 343, 269 and 320 m/s respectively, whereas the speed of sound in helium is approximately 1000 m/s. Furthermore, the gases in the current experiment were mixed with air in relatively low concentrations at the time when rats were responding. While this may have had a minor effect on U S V characteristics, it is unlikely that density alone was responsible for the increased number of calls detected during CO2 exposure. Britt's (1987) conclusion that rats experience distress during CO2 exposure was based partly on the occurrence of shaking, but this behaviour was not seen in our study. Britt (1987) did not state the flow rates used, but higher flow rates tend to result in a higher concentration of CO2 in the chamber at the time of loss of consciousness (Ambrose et al., 2000) and increase the likelihood that rats experience pain. Previous studies have also found increases in urination and defection during exposure to C 0  2  (Britt, 1987; Smith & Harrap, 1997). We did not record  urination and defecation as they often occurred when the animal was initially placed in the  51  c h a m b e r , and the l i k e l i h o o d o f future events is strongly related to the t i m e since these last occurred! T h e potential for a n i m a l s to experience distress d u r i n g euthanasia is l i m i t e d to the p e r i o d o f c o n s c i o u s n e s s , and i n this e x p e r i m e n t w e have a s s u m e d that c o m p l e t e loss o f consciousness o c c u r r e d w h e n the a n i m a l s b e c a m e f u l l y recumbent. R e c u m b e n c y i n rats d u r i n g CO2 exposure is associated w i t h a loss o f p e d a l and c o r n e a l reflexes (Hornett and H a y n e s , 1984) and w i t h a drop i n heart rate and onset o f an aberrant E E G ( C o e n e n et a l . , 1995). H o w e v e r , some researchers have f o u n d a delay b e t w e e n c o l l a p s e and loss o f reflexes ( D a n n e m a n et a l . , 1 9 9 7 ; H e w e t t et a l . , 1993), so it i s p o s s i b l e that the depth o f u n c o n s c i o u s n e s s varies at the t i m e o f r e c u m b e n c y . W e f o u n d that d u r i n g g r a d u a l - f i l l CO2 exposure, rats b e c a m e r e c u m b e n t after an average o f 106 s, a n d this f i n d i n g is consistent w i t h other studies u s i n g s i m i l a r f l o w rates ( H e w e t t et a l . , 1 9 9 3 ; H o r n e t t and H a y n e s , 1 9 8 4 ; D a n n e m a n et a l . , 1997). A n assessment o f a t a x i a w o u l d h a v e p r o v i d e d a n i n d i c a t i o n o f the t i m e that loss o f consciousness b e g a n to o c c u r , but w e were unable to accurately detect a t a x i a i n the current study because a loss o f m u s c l e c o o r d i n a t i o n c a n o n l y be detected w h e n particular postures and m o v e m e n t s are i n i t i a l l y present. F o r e x a m p l e , w h e n a rat is c r o u c h e d a n d stationary a r e d u c t i o n i n m u s c l e c o o r d i n a t i o n m a y not be o b v i o u s . In the current e x p e r i m e n t , s o m e rats e x h i b i t e d r e c u m b e n c y w i t h o u t o b v i o u s a t a x i a b e f o r e h a n d . T w o p o s s i b l e reasons for the rats' b e h a v i o u r a l response to CO2 are p a i n f r o m c a r b o n i c a c i d f o r m a t i o n at the m u c o s a and c o r n e a , and d y s p n e a f r o m h y p e r c a p n i a a n d h y p o x i a . W e f o u n d that the  O2 a n d CO2  concentrations  i n the c h a m b e r at the t i m e o f r e c u m b e n c y  were  a p p r o x i m a t e l y 1 4 % a n d 3 3 % . H o w e v e r , the O2 and CO2 concentrations were a p p r o x i m a t e l y 20%o a n d 5 % w h e n a n i m a l s started to r e s p o n d , and 1 5 % and 2 8 % w h e n a l l behaviours h a d p e a k e d a n d w e r e d e c l i n i n g . P h y s i o l o g i c a l data suggest that the CO2 t h r e s h o l d for the m a j o r i t y o f n o c i c e p t o r s i n rat nasal m u c o s a is b e t w e e n 3 7 a n d 5 0 % ( P e p p e l & A n t o n , 1993). H u m a n selfreport data indicate that CO2 is p a i n f u l i f a p p l i e d to the nasal m u c o s a at concentrations above  5  2  ,  •  4 7 % , a l t h o u g h this v a l u e ranges f r o m 32.5%  to 5 5 % d e p e n d i n g o n the i n d i v i d u a l ( A n t o n et a l . ,  1992). F o r the c o r n e a , h u m a n s report s t i n g i n g at 33%  and overt p a i n at 4 7 % CO2 ( C h e n et a l ,  1995). R e c e p t o r s f o u n d i n the l a r y n x , trachea, and b r o n c h i are also sensitive to i n h a l e d irritants ( r e v i e w e d i n W i d d i c o m b e , 2 0 0 1 ) , but responses to CO2 have not been f u l l y investigated for these receptors. H o w e v e r , D a n n e m a n et a l . (1997) had h u m a n subjects i n h a l e CO2 into the l o w e r a i r w a y s and f o u n d that o n l y 7 o f the 4 0 subjects reported that 5 0 % CO2 w a s o v e r t l y p a i n f u l , suggesting  that p a i n i n the  lower  airways  is not  occurring  at a m a r k e d l y  lower  CO2  c o n c e n t r a t i o n t h a n has been demonstrated for the nasal m u c o s a . A l t h o u g h it is p o s s i b l e that rats i n this study e x p e r i e n c e d s o m e p a i n before l o s i n g c o n s c i o u s n e s s , the CO2 concentration d u r i n g the p e r i o d o f m a x i m a l response w a s m u c h l o w e r than the p r o b a b l e p a i n t h r e s h o l d , and the rats d i d not e x h i b i t increases i n h e a d - s h a k i n g and f a c e - w a s h i n g . H e a d - s h a k i n g and f a c e - w a s h i n g have been o b s e r v e d i n rats d u r i n g exposure to moderate to h i g h CO2 concentrations ( B r i t t , 1 9 8 7 ; L e a c h et a l . , 2 0 0 2 ) , a n d f a c e - w a s h i n g has also been observed i n rats d u r i n g exposure to irritating c o m p o u n d s s u c h as c h l o r o f o r m ( B l a c k s a w et a l . , 1988). H e n c e , p a i n is u n l i k e l y to be the cause o f the rats' responses i n the current study. In contrast, the concentrations o f CO2 i n the c h a m b e r d u r i n g the p e r i o d o f m a x i m a l response have been f o u n d to cause sensations o f d y s p n e a i n spontaneously breathing h u m a n s ( D r i p p s & C o m r o e , 1 9 4 7 ; L i o t t i et a l . , 2 0 0 1 ) , and m a y cause s i m i l a r effect i n rodents. humans, hypercapnia  is also associated w i t h  other  negative  physical  symptoms  In  s u c h as  headache, f l u s h , restlessness, heart p o u n d i n g , d r o w s i n e s s a n d d i z z i n e s s ( M o o s a v i et a l . , 2 0 0 3 ) . It therefore seems l i k e l y that the distress response to g r a d u a l - f i l l CO2 exposure is due to d y s p n e a a n d other s y m p t o m s o f h y p e r c a p n i a rather than p a i n . F u r t h e r m o r e , D r i p p s and C o m r o e (1947) f o u n d that h u m a n s v a r y i n their response to h y p e r c a p n i a , a f i n d i n g consistent w i t h the v a r i a b i l i t y i n the rats' responses to CO2 exposure i n the current study. P r e v i o u s studies have assessed b r e a t h i n g i n rats d u r i n g CO2 exposure and f o u n d that it causes changes described as " l a b o u r e d 53  b r e a t h i n g " ( I w a r s s o n & R e h b i n d e r , 1993), " g a s p i n g a n d a s p h y x i a " ( C o e n e n et a l . , 1995) and " g a s p i n g or l a b o u r e d b r e a t h i n g " ( S m i t h & H a r r a p , 1997) p r i o r to loss o f consciousness, and these changes m i g h t be i n d i c a t i v e o f the sensation o f dyspnea. In the current study w e d i d not assess changes i n breathing because w e c o u l d not m a k e an accurate assessment f r o m v i d e o . I d e a l l y breathing c o u l d be assessed o b j e c t i v e l y b y m e a s u r i n g its f r e q u e n c y and depth, but this w a s b e y o n d the scope o f the current study. H o w e v e r , h u m a n m e d i c a l studies suggest that l a b o u r e d breathing is p o o r l y correlated w i t h the sensation o f d y s p n e a ( L u s h et a l . , 1988), so it is not clear that breathing measures are u s e f u l as an i n d i c a t o r o f d y s p n e a d u r i n g CO2 exposure. O u r results indicate that O2 r e d u c t i o n alone causes o n l y m i n i m a l distress i n rats o v e r the range o f O2 concentrations that w e e x a m i n e d . A l t h o u g h rats e x h i b i t e d a slight increase i n t o u c h i n g the nose to the l i d d u r i n g argon treatment, the increase i n this b e h a v i o u r was m u c h less than that seen i n C 0 2 - e x p o s e d a n i m a l s and there were no other b e h a v i o u r a l changes. T h i s result is consistent w i t h h u m a n self-report data suggesting that O2 levels i n the range seen d u r i n g this experiment  do not cause d y s p n e a d u r i n g  spontaneous breathing ( M o o s a v i  H o w e v e r , h y p e r c a p n i a and h y p o x i a are k n o w n to have synergistic  et a l . , 2 0 0 3 ) .  effects o n  responses ( N i e l s o n & S m i t h , 1952), s u c h that the response to increased C 0  2  ventilatory  m a y be potentiated  b y a l e v e l o f O2 r e d u c t i o n that has no effect o n its o w n . M a s u d a et a l . (2001) f o u n d that i n c r e a s i n g levels o f h y p o x i a augment the effect o f h y p e r c a p n i a o n d y s p n e a scores i n humans. A s s u m i n g that these results are a p p l i c a b l e to rats, it w o u l d appear that h y p o x i a d u r i n g CO2 euthanasia  may  increase  dyspnea,  and  this  is  supported  by  results  showing  that  O2  s u p p l e m e n t a t i o n reduces the adverse effects o f CO2 exposure o n rats ( C o e n e n et a l . , 1995). It is important to note that these results have no b e a r i n g o n distress associated w i t h argon w h e n used for euthanasia. R a t s c a n s u r v i v e for greater than 2 0 m i n u t e s w h e n e x p o s e d to O2 concentrations o f 4 . 9 % i n argon ( A l t l a n d et a l . , 1968), and our O2 concentrations w e r e o n l y reduced to 1 4 % . F o r t i m e l y u n c o n s c i o u s n e s s and death o f pigs and p o u l t r y w i t h a r g o n , concentrations o f 9 0 % or 54  greater have been used to reduce 0  2  levels below 2% (e.g., Raj, 1999; Raj et al., 1998), and  gradual fill exposure has not been investigated. Our study indicates that gradual-fill C 0 euthanasia causes distress in rats, and the 2  concentrations involved suggest that this distress is due to dyspnea rather than pain. The lack of consistency between experiments suggests that further research is necessary to examine whether there are strain differences in sensitivity or responsiveness to C 0 . Further research is also 2  necessary to determine the extent of the distress caused by C 0 exposure, and to determine 2  whether other gas euthanasia agents cause less distress. The variability in behavioural responses to C 0 suggests that tests of motivation might be a better approach. For example, approach2  avoidance testing could be used to examine whether rats will forgo an attractive reward of known value to avoid exposure to C 0 and other gas euthanasia agents. 2  55  Table 2.1. Descriptions of rat behaviours recorded during baseline and during exposure to CO2 or reduced O2 concentrations. Behaviour  Description  Activity  Movement that results in the back feet crossing a line that divides the length of the chamber in half (event).  Rear  Raising of the upper body while standing on the two back feet. Includes wall climbing. Climbing on the air sampling tube while chewing it and rearing during grooming were excluded (event).  Nose to lid  Time spent with the nose in contact with the chamber lid (state).  Escape behaviours: Scratch at lid  A rapid movement of the front paw from the lid through at least a 9 0 ° downward angle (event).  Push at lid  A  push at the chamber lid using the nose or front paw  evidenced by body and lid movement (event). Head shake  Rapid rotation of the head about the axis (event).  Face washing  Placement of one or both paws to the nose. Performance during grooming was excluded (event).  56  Table 2.2. Difference from baseline for each of the five behavioural responses of rats during air and C O 2 exposure (n = 8 rats). Data are presented as medians with 25 and 75 percentiles, and th  th  statistical comparisons were made with Wilcoxon Signed Ranks Test (T; based on N values >0). Air Behaviour Activity (no.) Rears (no.) Nose to lid (s) Escape behaviours (no.) Vocalizations (no.)  C0  2  T(N)  P-value  3.5 (2.0, 4.5)  0(8)  <0.005  0.0 (-2.5, 1.0)  10.5 (8.0,13.0)  0(8)  <0.005  0.0 (-15,6.5)  23.0 (9.0,25.5).  3(7)  <0.05  0.0 (0.0, 0.0)  4.0(0.5,10.5)  0(7)  <0.05  -0.5 (-1.0, 0.0)  6.0 (-0.5,13.0)  1.5 (7)  <0.05  Med (25 , 75 )  Med (25 , 75 )  0.0 (-0.5, 0.5)  m  th  th  57  th  "  Table 2.3. D i f f e r e n c e f r o m baseline f o r e a c h o f the f i v e b e h a v i o u r a l responses o f rats d u r i n g a i r and exposure to reduced O2 concentrations (n = 8 rats). D a t a are presented as m e d i a n s w i t h 2 5 t h and 7 5 t h percentiles, and statistical c o m p a r i s o n s were m a d e w i t h the W i l c o x o n S i g n e d R a n k s Test (T; based o n N v a l u e s >0).  Air Behaviour  A c t i v i t y (no.) R e a r s (no.)  N o s e to l i d (s) E s c a p e b e h a v i o u r s (no.) V o c a l i z a t i o n s (no.)  R e d u c e d O2  Med ( 2 5 , 75 ) t h  Med ( 2 5 , 75 )  th  t h  th  T  ^  /  >  V  a  ,  U  0.0 ( - 0 . 5 , 0 . 5 )  0.0(0.0,1.5)  5(8)  NS  -0.5 ( - 2 . 5 , 0 . 0 )  3.0(0.0,6.0)  4(7)  NS  0.0 ( - 3 . 5 , 0 . 0 )  2.5(1.0,8.0)  1.5(8)  <0.05  . 0 (0.0,0.0)  0.0(0.0,0.0)  0.0 ( - 0 . 5 , 0 . 0 )  0.0(0.0,0.5)  5(7)  NS  0  N S signifies P > 0.05  58  e  100 90 80 -  c o ro i_  70  50  c o o  40  V)  CD  o  5cm  O2  - • - 15cm 0  2  - • - 5cm C 0  2  -•- 15cm C 0  2  60  "c  CD O  -A-  30 20 10  0 120  60  180  240  300  360  420  480  Time (s) F i g u r e 2.1. Average concentrations o f 0  2  (open markers) and CO2 (filled markers) in the  chamber during the first 600 s o f the filling process. Concentrations were taken 5 cm (triangles) and 15 cm (squares) from the chamber bottom.  j  59  a) Activity  b) Rears  Time (s)  Time (s) d) Escape behaviours  c) Nose to lid  o <o o <o oto o) AS. cp y co  i? £> in P "o o <o o oI> f /x CIO VI CIO *I -  <o o <o o <o o <o <- co V  CO  ^  05  o  <o  o  <o  o  <o O  <p  »-  CO  V  CO  K  o  Time (s)  Time (s)  O)  e) Vocalizations 1.2 "O  -I  1-  0 0.8 -  *k_ cu Q. ^ »  O  z  0.6 0.4 0.2 -  0CO  to  o <o o Co  Time (s)  Figure 2.2. Responses by rats during the baseline period and then during exposure to either air (filled squares) or C O 2 (open squares) starting at t = 0. Median values per 15 s period are shown for a) activity, b) rears , c) nose to lid, d) escape behaviours, and e) vocalizations (n = 8 rats).  60  2.5 References A l t l a n d , P . D . , B r u b a c h , H . F . , P a r k e r , M . G . 1968. E f f e c t s o f inert gases o n tolerance o f rats to hypoxia. J. A p p l . Physiol. 24, 778-781. A m e r i c a n Veterinary M e d i c a l Association, 2001. 2000 Report  o f the A V M A  Panel  on  Euthanasia. J . A m . Vet, M e d . A s s o c . 2 1 8 , 669-696. A m b r o s e , N . , W a d h a m , J . , M o r t o n , D., 2 0 0 0 . R e f i n e m e n t i n E u t h a n a s i a . In: B a l l s , M . , v a n Z e l l e r , A . M . , H a i d e r , M . E . ( E d s ) , Progress i n the R e d u c t i o n , R e f i n e m e n t and R e p l a c e m e n t o f A n i m a l Experimentation, Elsevier Science, Amsterdam, pp.1159-1169. A n t o n , F., E u c h n e r , I., H a n d w e r k e r , H . O . , 1 9 9 2 . P s y c h o p h y s i c a l e x a m i n a t i o n o f p a i n i n d u c e d b y d e f i n e d CO2 pulses a p p l i e d to the nasal m u c o s a . P a i n 4 9 , 5 3 - 6 0 . A n t o n , F., P e p p e l , P., E u c h n e r , I.,  Handwerker, H.O.,  1991. Controlled noxious chemical  s t i m u l a t i o n : responses o f rat t r i g e m i n a l b r a i n s t e m neurones to CO2 pulses a p p l i e d to the nasal m u c o s a . N e u r o s c i . Lett. 1 2 3 , 2 0 8 - 2 1 1 . A u s t r a l i a n a n d N e w Z e a l a n d C o u n c i l for the C a r e o f A n i m a l s i n R e s e a r c h a n d T e a c h i n g , 1993. E u t h a n a s i a o f A n i m a l s U s e d for S c i e n t i f i c Purposes G l e n O s m o n d ,  ANZCCART.  B a n z e t t , R . B . , L a n s i n g , R . W . , E v a n s , K . C . , S h e a , S . A . 1996. S t i m u l u s - r e s p o n s e characteristics o f C 0 2 - i n d u c e d air hunger i n n o r m a l subjects. R e s p . P h y s i o l . 1 0 3 , 1 9 - 3 1 . B l a c k s h a w , J . K . , F e n w i c k , D . C . , Beattie, A . W . , A l l a n , D . J . , 1988. T h e b e h a v i o u r o f c h i c k e n s , m i c e and rats d u r i n g euthanasia w i t h c h l o r o f o r m , c a r b o n d i o x i d e and ether. L a b . A n i m . 2 2 , 67-75.  '  Blanchard, R.J., Y u d k o , E.B., Blanchard, D.C., Taukulis, H.K.,  1993. High-frequency (35-70  k H z ) ultrasonic v o c a l i z a t i o n s i n rats confronted w i t h anesthetized c o n s p e c i f i c s : effects o f gepirone, ethanol, and d i a z e p a m . P h a r m . B i o c h e m . B e h a v . 4 4 , 3 1 3 - 3 1 9 . B r i t t , D . P., 1987. T h e humaneness o f c a r b o n d i o x i d e as a n agent o f euthanasia for laboratory  61  rodents. In: E u t h a n a s i a o f U n w a n t e d , Injured or D i s e a s e d A n i m a l s or for E d u c a t i o n a l or Scientific Purposes, pp. 19-31. Potter's B a r : Universities Federation for A n i m a l Welfare. Burgdorf, J., Knutson, B.,  Panksepp, J. 2000. Anticipation of rewarding  electrical  brain  s t i m u l a t i o n e v o k e s ultrasonic v o c a l i z a t i o n s i n rats. B e h a v . N e u r o s c i . 114, 3 2 0 - 3 2 7 . C a n a d i a n C o u n c i l o n A n i m a l C a r e , 1 9 9 3 . G u i d e to the C a r e and U s e o f E x p e r i m e n t a l A n i m a l s , V o l u m e 1, 2  n d  Edition, Olfert, E.D., Cross, B . M . , M c W i l l i a m , A . A (Eds). Ottawa, C C A C .  C h e n , X . , Gallar, J., P o z o , M . A . , Baeza, M . , Belmonte, C , 1995. C 0 a comparison between  h u m a n sensation and nerve a c t i v i t y  2  s t i m u l a t i o n o f the c o r n e a :  in polymodal  nociceptive  afferents o f the cat. E u r . J . N e u r o s c i . 7, 1 1 5 4 - 1 1 6 3 . Close, B., Banister, K.,  Baumans, V.,  B e r n o t h , E., B r o m a g e , N . , B u n y a n , J . , Erhardt,  F l e c k n e l l , P., G r e g o r y , N . , H a c k b a r t h , H . , Commission Working  Party Report:  M o r t o n , D.,  Warwick,  R e c o m m e n d a t i o n s for  C,  W.,  1997. European  euthanasia o f  experimental  a n i m a l s , Part I. L a b . A n i m . 3 0 , 2 9 3 - 3 1 6 . C o e n e n , A . M . , D r i n k e n b u r g , W . H . , H o e n d e r k e n , R., v a n L u i j t e l a a r , G . L . , 1995. C a r b o n d i o x i d e euthanasia i n rats: o x y g e n s u p p l e m e n t a t i o n m i n i m i z e s signs o f agitation and a s p h y x i a . L a b . A n i m . 29, 262-268. D a n n e m a n , P . J . , S t e i n , S . , W a l s h a w , S . O . , 1997. H u m a n e and p r a c t i c a l i m p l i c a t i o n s o f u s i n g c a r b o n d i o x i d e m i x e d w i t h o x y g e n f o r anesthesia or euthanasia o f rats. L a b . A n i m . S c i . 4 7 , 376-85. D r i p p s , R . D . , C o m r o e , J . H . , 1 9 4 7 . T h e respiratory and c i r c u l a t o r y response o f n o r m a l m a n to i n h a l a t i o n o f 7.6 a n d 10.4 per cent CO2 w i t h a c o m p a r i s o n o f the m a x i m a l v e n t i l a t i o n produced  by  severe  muscular  exercise,  inhalation  of  CO2  and  maximal  voluntary  hyperventilation. A m . J. P h y s i o l . 149, 4 3 - 5 1 . F e n g , Y . , S i m p s o n , T. L., 2 0 0 3 . N o c i c e p t i v e sensation and sensitivity e v o k e d f r o m h u m a n c o r n e a and c o n j u n c t i v a s t i m u l a t e d b y CO2. Invest. O p h t h . V i s . S c i . 4 4 , 5 2 9 - 5 3 2 . 62  H a c k b a r t h , H . , K u p p e r s , N . , B o h n e t , W . , 2 0 0 0 . E u t h a n a s i a o f rats w i t h c a r b o n d i o x i d e — a n i m a l w e l f a r e aspects. L a b . A n i m . 3 4 , 9 1 - 9 6 . H e w e t t , T . A . , K o v a c s , M . S . , A r t w o h l , J . E . , Bennett, B . T . ,  1 9 9 3 . A c o m p a r i s o n o f euthanasia  methods i n rats, u s i n g c a r b o n d i o x i d e i n p r e - f i l l e d and f i x e d f l o w rate f i l l e d chambers. L a b . A n i m . Sci. 43, 579-582. Hornett, T . D . , H a y n e s , A . R . , 1984. C o m p a r i s o n o f c a r b o n dioxide/air m i x t u r e and nitrogen/air m i x t u r e for the euthanasia o f rodents. D e s i g n o f a system for i n h a l a t i o n euthanasia. A n i m a l Technology 35, 93-99. Iwarsson , K., Rehbinder, C ,  1 9 9 3 . A study o f different euthanasia techniques i n g u i n e a p i g s ,  rats, a n d m i c e . A n i m a l response and p o s t m o r t e m f i n d i n g s . S c a n . J . L a b . A n i m . S c i . 2 0 , 1 9 1 205. Knutson, B., Burgdorf,  J., Panksepp, J.  1998. A n t i c i p a t i o n o f p l a y elicits  high-frequency  ultrasonic v o c a l i z a t i o n s i n y o u n g rats. J . C o m p . P s y c h o l . 1 1 2 , 6 5 - 7 3 . K n u t s o n , B., Burgdorf, J . , Panksepp, J. 1999. High-frequency  ultrasonic vocalizations  index  c o n d i t i o n e d p h a r m a c o l o g i c a l r e w a r d i n rats. P h y s i o l . B e h a v . 6 6 , 6 3 9 - 6 4 3 . K n u t s o n , B . , B u r g d o r f , J . , P a n k s e p p , J . , 2 0 0 2 . U l t r a s o n i c v o c a l i z a t i o n s as i n d i c e s o f affective states i n rats. P s y c h o l . B u l l . 1 2 8 , 9 6 1 - 9 7 7 . L a n s i n g , R . W . , I m , B . S . H . , T h w i n g , J.I., L e g e d z a , A . T . R . , B a n z e t t , R . B . 2 0 0 0 . T h e p e r c e p t i o n o f respiratory w o r k and effort c a n be independent o f the p e r c e p t i o n o f air hunger. A m . J . Respir. Crit. Care M e d . 162, 1690-1696. L e a c h , M . C , B o w e l l , V . A . , A l l a n , T . F . , M o r t o n , D . B . , 2 0 0 2 . A v e r s i o n to gaseous euthanasia agents i n rats and m i c e . C o m p a r a t i v e M e d . 5 2 , 2 4 9 - 2 5 7 . Liotti, M . , Brannan, S., Egan, G.,  Shade, R.,  M a d d e n , L.,  Abplanalp, B., Robillard,  R.,  L a n c a s t e r , J . , Z a m a r r i p a , F . E . , F o x , P.T., D e n t o n , D . , 2 0 0 1 . B r a i n responses associated w i t h c o n s c i o u s n e s s o f breathlessness (air hunger). P r o c . N a t . A c a d . S c i . 9 8 , 2 0 3 5 - 2 0 4 0 . 63  L u s h , M . T . , J a n s o n - B j e r k l i e , S . , C a r r i e r i , V . K . , L o v e j o y , N . 1 9 8 8 . D y s p n e a i n the ventilatorassisted patient. Heart a n d L u n g , 17, 5 2 8 - 5 3 5 . M a s u d a , A . , O h y a b u , Y . , K o b a y a s h i , T., Y o s h i n o , C , S a k a k i b a r a , Y . , K o m a t s u , T., H o n d a , Y . , 2 0 0 1 . L a c k o f p o s i t i v e interaction b e t w e e n CO2 and h y p o x i c s t i m u l a t i o n for Pco2 -  VAS  response slope i n h u m a n s . R e s p . P h y s i o l . 126, 1 7 3 - 1 8 1 . M o o s a v i , S . H . , G o l e s t a n i a n , E . , B i n k s , A . P . , L a n s i n g , R . W . , B r o w n , R., B a n z e t t , R . B . , 2 0 0 3 . H y p o x i c a n d h y p e r c a p n i c drives to breathe generate equivalent l e v e l s o f air hunger  in  humans. J. A p p l . Physiol. 94, 141-154. N i e l s o n , M . , S m i t h , H . , 1 9 5 2 . Studies o n the r e g u l a t i o n o f respiration i n acute h y p o x i a . A c t a . Physiol. Scand. 24, 293-313. P e p p e l , P., A n t o n , F., 1 9 9 3 . R e sp o nses o f rat m e d u l l a r y d o r s a l h o r n neurons f o l l o w i n g intranasal noxious  c h e m i c a l s t i m u l a t i o n : effects o f s t i m u l u s intensity, d u r a t i o n , and interstimulus  interval. J . N e u r o p h y s i o l . 7 0 , 2 2 6 0 - 2 2 7 5 . R a j , A . B . M . , 1 9 9 9 . B e h a v i o u r o f p i g s e x p o s e d to m i x t u r e s o f gases and the t i m e required to stun and k i l l t h e m : w e l f a r e i m p l i c a t i o n s . V e t . R e c o r d 144, 1 6 5 - 1 6 8 . R a j , A . B . M . , W o t t o n , S . B . , M c K i n s t r y , J . L . , H i l l e b r a n d , S . J . W . , Pieterse, C , 1 9 9 8 . C h a n g e s i n the somatosensory  e v o k e d potentials and spontaneous  electroencephalogram o f  broiler  c h i c k e n s d u r i n g exposure to gas m i x t u r e s . B r i t . P o u l t r y S c i . 3 9 , 6 8 6 - 6 9 5 . R o b e r t s , L . H . , 1 9 7 5 . T h e rodent ultrasound p r o d u c t i o n m e c h a n i s m . U l t r a s o n i c s 1 3 , 8 3 - 8 8 . S a l e s , G . D . 1 9 7 2 . U l t r a s o u n d and aggressive b e h a v i o u r i n rats and other s m a l l m a m m a l s . A n i m . Behav. 20, 88-100. Sanders, I., W e i s z , D . J . , Y a n g , B . Y . , F u n g , K . , A m i r a l i , A . , 2 0 0 1 . T h e m e c h a n i s m o f ultrasonic v o c a l i z a t i o n i n the rat. S o c . N e u r o s c i . A b s t r . , V o l . 2 7 , P r o g r a m N o . 8 8 . 1 9 . S h e a , S . A . , H a r t y , H . R . , B a n z e t t , R . B . , 1996. S e l f - c o n t r o l o f m e c h a n i c a l v e n t i l a t i o n to m i n i m i z e CO2 i n d u c e d air hunger. R e s p . P h y s i o l . 1 0 3 , 1 1 3 - 1 2 5 . 64  S m i t h , W . , H a r r a p , S B . , 1 9 9 7 . B e h a v i o u r a l a n d c a r d i o v a s c u l a r responses o f rats to euthanasia u s i n g c a r b o n d i o x i d e gas. L a b . A n i m . 3 1 , 3 3 7 - 3 4 6 . T h o m a s , D . A . , T a k a h a s h i , L . K . , B a r f i e l d , R . J . 1 9 8 3 . A n a l y s i s o f ultrasonic v o c a l i z a t i o n s emitted b y intruders d u r i n g aggressive encounters a m o n g rats (Rattus norvegicus). J . C o m p . P s y c h o l . 97,201-206. U n i t e d K i n g d o m H o m e O f f i c e , 1997. T h e H u m a n e K i l l i n g o f A n i m a l s under S c h e d u l e 1 to the Animals  (Scientific  Procedures)  A c t 1986 C o d e  o f Practice. N o r w i c h ,  Stationery O f f i c e . W i d d i c o m b e , J . , 2 0 0 1 . A i r w a y receptors. R e s p . P h y s i o l . 1 2 5 , 3 - 1 5 .  65  H e r Majesty's  CHAPTER 3: Rats avoid exposure to carbon dioxide and argon  3  3.1 Introduction Small laboratory rodents are euthanized using a number of methods, including physical techniques, injectable anaesthetics, and exposure to volatile anaesthetics and other gases. One of the most common methods is exposure to CO2. Animals are exposed to either a gradually increasing concentration of CO2 or a pre-filled chamber, and this causes unconsciousness followed by death. Ideally, a euthanasia method should result in a quick death with minimal pain and distress. In humans CO2 is known to cause dyspnea, a sensation of breathlessness, at concentrations of 8% (Dripps & Comroe, 1947; Liotti et al., 2001). At CO2 concentrations ranging from 30% to 54%, humans also experience pain at the cornea (Chen et al., 1995; Feng & Simpson, 2003), conjunctiva (Feng & Simpson, 2003) and nasal mucosa (Anton et al., 1992; ,Thurauf et al., 2002). In rats, the threshold for the majority of nociceptors in the nasal mucosa is between 37% and 50%> C 0 (Anton et al., 1991; Peppel & Anton, 1993). Some studies have 2  reported no behavioural evidence of distress in rats during pre-fill (Blackshaw et al., 1988; Smith & Harrap, 1997) and gradual-fill C 0 exposure (Hackbarth et al., 2000; Hornett & 2  Haynes, 1984; Smith & Harrap, 1997). However, others have suggested that CO2 causes behavioural signs of distress (Britt, 1987; Coenen et al., 1995; Iwarsson & Rehbinder, 1993; Chapter 2). This variability between experiments suggests that simply monitoring behavioural responses during exposure may be inadequate as a method for assessing the rat's perception of C0 . 2  A version of this chapter has been accepted for publication. Niel, L., Weary, D . M . , 2006. Rats avoid exposure to carbon dioxide and argon. Appl. Anim. Behav. Sci. (accepted). 3  66  A v e r s i o n to CO2 exposure has also b e e n e x a m i n e d u s i n g preference testing. L e a c h et a l . (2002) f o u n d that rats m o v e d to a n a i r f i l l e d c h a m b e r w h e n e x p o s e d to moderate CO2 concentrations. H o w e v e r , o n l y static concentrations between 2 5 . 5 % a n d 50.8%) were tested. R a t s ' responses to l o w e r concentrations a n d to g r a d u a l - f i l l exposure have not been e x a m i n e d , and to date no studies have addressed the strength o f a v e r s i o n to CO2. A n o t h e r f o r m o f preference testing, the a p p r o a c h - a v o i d a n c e test, has been u s e d to e x a m i n e a v e r s i o n to CO2 i n m i n k ( C o o p e r et a l . , 1998), p i g s ( R a j & G r e g o r y , 1995) a n d p o u l t r y ( R a j , 1 9 9 6 ; G e r r i t z e n et a l . , 2 0 0 0 ; W e b s t e r & F l e t c h e r , 2 0 0 4 ) . D u r i n g this procedure, entry into the c h a m b e r is v o l u n t a r y a n d a n i m a l s are m o t i v a t e d to enter a n d r e m a i n f o r a r e w a r d . I f a n i m a l s a v o i d the c h a m b e r , e v e n w h e n it contains s o m e t h i n g that they are trained a n d m o t i v a t e d to o b t a i n s u c h as a f o o d r e w a r d , this indicates that they find the c o n d i t i o n s o f the test cage a v e r s i v e . A p p r o a c h - a v o i d a n c e testing has not p r e v i o u s l y b e e n u s e d to e x a m i n e a v e r s i o n to CO2 i n rats. T h e a i m o f this study w a s to use a p p r o a c h - a v o i d a n c e testing to characterize rats' a v e r s i o n to static a n d g r a d u a l - f i l l CO2 exposure. W e also e x a m i n e d whether rats  exhibit  a v e r s i o n to 9 0 % argon i n air, w h i c h causes death b y r e d u c i n g O2 l e v e l s to 2% a n d has b e e n p r o p o s e d as a n alternative m e t h o d o f gas euthanasia f o r rats ( L e a c h et a l . , 2 0 0 2 ) .  3.2 Materials and Methods 3.2.1 Subjects a n d H o u s i n g T h e subjects w e r e 10 m a l e W i s t a r rats, 4 0 0 to 5 0 0 g , o b t a i n e d f r o m the U B C A n i m a l C a r e C e n t r e R o d e n t B r e e d i n g U n i t as surplus s u p p l y stock a n d destined f o r euthanasia. A n i m a l r o o m s were kept at 21 ± 1 °C under a 1 2 : 1 2 - h r l i g h t - d a r k c y c l e , a n d rats were g i v e n a d l i b i t u m  67  access to f o o d ( L a b D i e t 5 0 0 1 , P M I N u t r i t i o n International, R i c h m o n d , U S A ) and tap water. A l l testing w a s c o n d u c t e d d u r i n g the l i g h t p o r t i o n o f the l i g h t - d a r k c y c l e . R a t s were s i n g l y h o u s e d i n the testing apparatus, c o n s i s t i n g o f t w o transparent cages c o n n e c t e d b y a n opaque t u n n e l m a d e o f b l a c k , r i b b e d , P V C t u b i n g w i t h a diameter o f 10 c m a n d s l o p e d so that one cage w a s 2 7 c m higher t h a n the other. T h e ' h o m e ' cage m e a s u r e d 48 x 38 x 2 0 c m , a n d c o n t a i n e d f o o d , water, b e d d i n g , an opaque nestbox and a N y l a b o n e d o g c h e w . T h e secondary cage m e a s u r e d 4 5 x 2 4 x 2 0 c m and contained b e d d i n g . T h e b o t t o m cage w a s a l w a y s u s e d for testing because the test gases were denser than air. D u r i n g p r e l i m i n a r y testing w e d e t e r m i n e d that the test gases were restricted to the b o t t o m cage a n d the l o w e r p o r t i o n o f the t u n n e l . T o determine the effect o f cage f a m i l i a r i t y , h a l f o f the rats were tested i n the h o m e cage and the other h a l f w e r e tested i n the secondary cage. T h i s w a s a c c o m p l i s h e d b y p o s i t i o n i n g the h o m e cage o n the b o t t o m for h a l f o f the rats, and the secondary cage o n the b o t t o m for the other h a l f o f the rats. H o w e v e r , m a n y rats spent a p o r t i o n o f their t i m e i n the secondary cage, so were f a m i l i a r w i t h b o t h cages.  3.2.2 T e s t i n g P r o c e d u r e D u r i n g e x p e r i m e n t a l testing, each a n i m a l and its testing apparatus w e r e  transferred  i n d i v i d u a l l y to a test r o o m . A t this t i m e , the nest b o x w a s r e m o v e d and the w i r e l i d o n the test cage w a s r e p l a c e d w i t h a p l e x i g l a s l i d that featured t w o air outlets p o s i t i o n e d at the end closest to the t u n n e l , a gas inlet at the far end o f the test cage, a n d a gas s a m p l i n g tube inserted at the center o f the test cage. T h e air outlets were c o v e r e d w i t h m e s h to prevent the rats f r o m p u s h i n g their noses outside the c h a m b e r . T h e experimenter w a s c o n c e a l e d b e h i n d a b l i n d d u r i n g testing. T h e testing apparatus a n d 0  2  meter readout were v i d e o r e c o r d e d d u r i n g testing.  A i r , C O 2 and a r g o n were d e l i v e r e d to the test cage f r o m c o m p r e s s e d gas c y l i n d e r s ( P r a x a i r , R i c h m o n d , B . C . ) . T h e treatment gases were passed t h r o u g h a copper c o i l i n a r o o m 68  temperature water bath to regulate gas temperature before entering the test cage. F l o w rates o f gases were m e a s u r e d u s i n g a v a r i a b l e area f l o w meter ( D w y e r Instruments V S B - 6 6 - B V ) , and measured f l o w rates for CO2 were adjusted for density u s i n g a c o r r e c t i o n factor o f 0 . 8 1 2 . O2 concentrations i n the test cage were m o n i t o r e d d u r i n g the e x p e r i m e n t u s i n g a M o c o n L F 7 0 0 D 0  2  analyzer, and were u s e d to calculate CO2 concentrations at s p e c i f i c t i m e points (t = x ) w i t h  the f o r m u l a : C 0  2 ( 1  =  x )  = 100 - (100 *  ( [ 0 , = ] / [ 0 (, = <,)])). 2{  x)  2  T e s t i n g o f the apparatus w a s c o m p l e t e d to ensure that CO2 concentrations were u n i f o r m throughout the test cage d u r i n g g r a d u a l - f i l l CO2 a d d i t i o n . D u r i n g CO2 a d d i t i o n at a rate o f 1 7 % o f the test cage v o l u m e per m i n u t e , gas concentrations were m o n i t o r e d at a depth o f h a l f the test cage height at f i v e different sites. T h e r e were n o o b v i o u s trends for l o w e r CO2 concentrations at the end o f the c h a m b e r closest to the t u n n e l , and CO2 concentrations at the different sites i n the c h a m b e r v a r i e d b y less than 3 % . B e f o r e b e g i n n i n g the e x p e r i m e n t , rats were trained to p e r f o r m the a p p r o a c h - a v o i d a n c e task. R a t s were trained for 10 days w i t h air o n l y and this w a s f o l l o w e d b y 9 days o f t r a i n i n g where air and different concentrations o f CO2 were alternated to f a m i l i a r i z e the a n i m a l s w i t h gas exposure and r e m o v e any effects o f n o v e l t y . F o r this f i n a l stage o f t r a i n i n g a l l rats were e x p o s e d to air, g r a d u a l - f i l l CO2 at 1 7 % o f the test cage v o l u m e per m i n u t e and static CO2 concentrations o f 5 , 10, 15 a n d 2 0 % . A n i m a l s were not e x p o s e d to argon p r i o r to testing because w e were c o n c e r n e d that this w o u l d affect p e r f o r m a n c e i n general. In p r e l i m i n a r y testing one rat that w a s e x p o s e d to argon appeared to have d i f f i c u l t y d e t e r m i n i n g w h i c h cage c o n t a i n e d air and refused to r u n the task the f o l l o w i n g day. F u r t h e r m o r e , argon w a s not expected to e v o k e a n o v e l t y response because it is an odourless and n o n - i r r i t a t i n g gas. D u r i n g b o t h t r a i n i n g and e x p e r i m e n t a l sessions, rats were first l o c k e d into the top cage for 5 m i n to a l l o w t i m e for a d d i t i o n o f treatment gases a n d f o o d rewards to the test cage. F o l l o w i n g l o c k r e m o v a l they were able to enter the l o w e r test cage for a f o o d r e w a r d o f 2 0 69  H o n e y N u t C h e e r i o s ™ ( G e n e r a l M i l l s , Inc., M i n n e s o t a ) . F o r static e x p o s u r e , the test cage w a s p r e - f i l l e d w i t h either CO2 or argon. F o r gradual f i l l e x p o s u r e , CO2 flow into the test cage w a s initiated w h e n the rat started eating the f o o d r e w a r d . T h e session ended 3 0 0 s after l o c k r e m o v a l , a n d a n i m a l s were a l l o w e d to exit a n d re-enter the test cage d u r i n g this p e r i o d . A t the end o f the session, the r e m a i n i n g r e w a r d items were r e m o v e d and the rat w a s returned to the h o l d i n g r o o m . O n the final d a y o f t r a i n i n g , a l l rats were r u n w i t h air i n the test cage and p e r f o r m e d the task c o r r e c t l y , c o n s u m i n g at least 19 r e w a r d i t e m s each d u r i n g the first entry into the test cage. T h e e x p e r i m e n t consisted o f three test periods. T h e a i m o f Part 1 w a s to determine w h i c h static concentrations o f CO2 rats find aversive b y  filling  the test cage w i t h air or static CO2 at  concentrations o f 5 , 10, 15 a n d 2 0 % . E a c h rat w a s tested once w i t h each c o n d i t i o n over a 5 - d p e r i o d . Treatment  order  was  allocated a c c o r d i n g to a L a t i n square and  coun ter balanc ed  a c c o r d i n g to h o m e cage p o s i t i o n i n g ( h i g h vs. l o w ) . W e r e c o r d e d the total n u m b e r o f r e w a r d items eaten over the entire test session as w e l l as the eating and d w e l l i n g t i m e s for the first entry (the m a x i m u m exposure t i m e tolerated), a n d p r e d i c t e d that these variables w o u l d d e c l i n e w i t h i n c r e a s i n g CO2 concentration. W e also r e c o r d e d attempted entries into the test cage as a measure o f m o t i v a t i o n to enter the test cage w i t h each treatment. F i n a l l y , w e predicted that i f a treatment w a s a v e r s i v e , it w o u l d increase the t i m e t a k e n to enter the test cage o n the f o l l o w i n g day. T h e r e f o r e , w e r e c o r d e d the latency to enter the tube l e a d i n g to the test cage f o l l o w i n g l o c k removal. T h e a i m o f Part 2 w a s to determine what concentration o f CO2 rats find aversive d u r i n g g r a d u a l - f i l l e x p o s u r e , i n order to c o m p a r e it to the static fill data f r o m Part 1. Rats were g i v e n a single exposure to g r a d u a l - f i l l CO2 at a rate o f 17%) o f the test cage v o l u m e per m i n u t e . W e w e r e s p e c i f i c a l l y interested i n the gas concentrations w h e n the rats stopped eating and left the test cage so the O2 concentration w a s the o n l y v a r i a b l e r e c o r d e d . T h i s test t o o k place the day after the end o f testing for Part 1. 70  T h e a i m o f Part 3 w a s to evaluate rats' responses to a static c o n c e n t r a t i o n ( 9 0 % ) o f a r g o n gas i n air ( 2 % O2), a n d c o m p a r e this w i t h air exposure. T h i s test t o o k p l a c e the d a y after testing w i t h g r a d u a l - f i l l C O 2 exposure. A s described a b o v e , some adverse reactions to argon were o b s e r v e d d u r i n g p i l o t testing, a n d due to the potential f o r c a r r y o v e r effects f o l l o w i n g a r g o n e x p o s u r e , rats were e x p o s e d to air o n the first d a y a n d a r g o n o n the s e c o n d d a y . V a r i a b l e s r e c o r d e d were i d e n t i c a l to Part 1 except the treatments were n o t c o u n t e r b a l a n c e d , so the latency to enter the tube l e a d i n g t o the test cage w a s n o t recorded.  3.2.3 Statistical Analysis O n e a n i m a l d i d not learn the task a n d w a s r e m o v e d f r o m the e x p e r i m e n t . T h e data f o r Part 1 a n d Part 3 w e r e n o n - n o r m a l w i t h unequal variances a n d c o u l d not be corrected t h r o u g h the use o f transformations, so n o n - p a r a m e t r i c statistics were u s e d f o r a n a l y s i s . A n i n i t i a l e v a l u a t i o n o f the data i n d i c a t e d there were n o differences f o r a n y v a r i a b l e s between rats tested i n the h o m e cage versus the secondary cage, therefore the data were p o o l e d f o r further analysis. F o r Part 1, the F r i e d m a n ' s test w a s used to c o m p a r e differences i n dependant variables across static C O 2 concentrations o f 0 , 5 , 10, 15 and 2 0 % . F o r Part 2 , d e s c r i p t i v e statistics are presented o n l y . F o r Part 3 , the W i l c o x o n S i g n e d R a n k s test w a s used to c o m p a r e differences i n dependent v a r i a b l e s f o r a i r a n d a r g o n exposure.  3.3 Results 3.3.1 Part 1 - Exposure to static concentrations of CO2 A l l 9 rats entered the test cage at 0 , 5 , 10 a n d 1 5 % . C 0 . A t 2 0 % . C 0 o n e rat refused to 2  2  enter the test cage. A t 0 , 5 a n d 1 0 % C O 2 rats ate a l l o f the r e w a r d items p r o v i d e d , but the n u m b e r eaten d e c l i n e d w i t h 15 and 2 0 % C O 2 ( F i g . 3 . 1 a ; P < 0.005). A t 1 5 % one a n i m a l refused  71  to eat. A t 2 0 % CO2 o n l y t w o a n i m a l s ate, a n d e a c h c o n s u m e d o n l y o n e or t w o r e w a r d items. C o n s i d e r i n g o n l y the first entry into the test cage, the eating a n d d w e l l i n g t i m e s were r e d u c e d at the highest C 0  2  concentrations (for b o t h : P < 0.005). F r o m 0 % to 1 0 % C 0  2  these variables  d e c l i n e d o n l y s l i g h t l y ( F i g . 3.1b), but there w a s increased v a r i a b i l i t y i n response at 10%) C 0 . A t 2  15%> there w a s a steep drop i n eating a n d d w e l l i n g t i m e s ( m e d i a n 3 2 s a n d 4 6 s, r e s p e c t i v e l y ) , a n d at 2 0 % C 0 these values h a d d r o p p e d again ( m e d i a n 2 s a n d 5 s, respectively). T h e total ;  2  n u m b e r o f t i m e s that the rats attempted to enter the test cage d u r i n g the entire session d i f f e r e d across the f i v e C 0 concentrations ( F i g . 3 . 1 c ; P < 0.005). A t 0 a n d 5%> C 0 rats entered the test 2  2  cage o n l y once a n d r e m a i n e d f o r the m a j o r i t y o f the session, but at h i g h e r concentrations rats s h o w e d a n i n c r e a s i n g n u m b e r s o f entries. T h e t i m e t a k e n to enter the tube l e a d i n g to the test cage w a s n o t affected b y the treatment o n the p r e v i o u s d a y (P > 0.1). O n average rats t o o k 2.3 ± 2 s to enter the tube.  3.3.2 Part 2 - Gradual-fill C 0 exposure 2  During gradual-fill C 0  2  exposure, rats stopped eating a n d left the test cage at C 0  2  concentrations o f 17.3 ± 2.1% (mean ± standard deviation) and 18.4 ± 2.0% respectively.  3.3.3 Part 3 - Argon exposure D u r i n g a r g o n exposure, three rats refused to enter the test c a g e , n o rats ate, a n d the m e d i a n d w e l l i n g t i m e w a s 3 s ( T a b l e 3.1). T h e m e d i a n n u m b e r o f entries d u r i n g argon exposure w a s t w o . In contrast, d u r i n g ai r exposure rats ate a l l o f the r e w a r d items a n d spent almost the entire session i n the test cage. T h e n u m b e r o f r e w a r d items eaten, a n d the eating a n d d w e l l i n g times were a l l s i g n i f i c a n t l y greater d u r i n g the session w i t h air (P < 0 . 0 0 5 ) .  72  3.4 Discussion T h e u l t i m a t e a i m o f gas euthanasia is to d e l i v e r gases i n s u c h a w a y as to render the animal unconscious without  c a u s i n g distress. In the current  study rats tolerated  extended  exposure to 5 % and 1 0 % CO2, but this w a s not s u f f i c i e n t to cause u n c o n s c i o u s n e s s . T h e rats w e r e u n w i l l i n g to tolerate extended exposure to 1 5 % and 2 0 % CO2, and concentrations greater than 30%o are necessary to cause loss o f consciousness ( C h a p i n & E d g a r , 1 9 6 3 ; Chapter 2). N o c i c e p t o r s i n rat nasal m u c o s a b e g i n to respond to CO2 concentrations o f a p p r o x i m a t e l y 2 5 % ( A n t o n et a l . , 1 9 9 1 ; P e p p e l & A n t o n , 1993), so p a i n is u n l i k e l y to be the cause o f CO2 a v e r s i o n at the t w o highest concentrations that w e tested. D y s p n e a due to h y p e r c a p n i a m o r e l i k e l y accounts for our results. S o m e h u m a n s report d y s p n e a at CO2 l e v e l s o f o n l y 8 % ( D r i p p s & C o m r o e , 1 9 4 7 ; L i o t t i et a l . , 2 0 0 1 ) , a n d this sensation increases i n severity w i t h h i g h e r  CO2  concentrations. D y s p n e a c a n also be a c c o m p a n i e d b y other negative p h y s i c a l s y m p t o m s s u c h as headache, f l u s h , restlessness, heart p o u n d i n g , d r o w s i n e s s and d i z z i n e s s ( M o o s a v i et a l . , 2 0 0 3 ) . H o w e v e r , h u m a n s v a r y i n their tolerance to h y p e r c a p n i a ( D r i p p s & C o m r o e , 1947). S i m i l a r differences i n tolerance i n rats m i g h t e x p l a i n the v a r i a b i l i t y i n eating and d w e l l i n g t i m e s o b s e r v e d at 1 0 % CO2 i n the current study. T h e v a r i a b i l i t y at 1 0 % CO2 a n d the steep d r o p i n eating and d w e l l i n g t i m e s at 15%> CO2 suggests that the onset o f severe d y s p n e a i n rats m a y o c c u r w i t h 1 0 % to 1 5 % C 0 . 2  It  has been suggested that g r a d u a l - f i l l CO2  exposure  results i n a s l o w onset  of  u n c o n s c i o u s n e s s w i t h o u t distress, but i n the current study rats left the test cage w h e n the CO2 c o n c e n t r a t i o n reached o n average  1 8 . 4 % . T h i s CO2 concentration is consistent w i t h rats'  a v o i d a n c e o f static CO2 concentrations, suggesting that g r a d u a l - f i l l exposure does not cause a gradual loss o f consciousness w i t h o u t c a u s i n g a v e r s i o n . In other research w e have f o u n d that rats d o not b e c o m e r e c u m b e n t u n t i l a CO2 concentration o f a p p r o x i m a t e l y 3 0 % is reached, about  73  105 s after gas flow is initiated at a flow rate o f 1 7 % per m i n u t e (Chapter 2). T h e depth o f u n c o n s c i o u s n e s s at the onset o f r e c u m b e n c y is u n k n o w n since s o m e studies report a short delay before a loss o f reflexes occurs ( D a n n e m a n et a l . , 1 9 9 7 ; H e w e t t et a l . , 1993). T h i s suggests that s o m e awareness o f aversive CO2 concentrations m i g h t persist for a short p e r i o d after onset o f r e c u m b e n c y . In the present study, a concentration o f 1 8 % w a s reached after 6 0 s, suggesting that d u r i n g g r a d u a l - f i l l CO2 e x p o s u r e , rats are e x p o s e d to aversive CO2 concentrations for at least 45 s before  losing  consciousness.  Increased  flow  rates  would  expose  rats to  higher  CO2  concentrations, but l i k e l y for a shorter d u r a t i o n before u n c o n s c i o u s n e s s . T h e net effects o n the rats o f this c o n f l i c t b e t w e e n intensity and d u r a t i o n are u n k n o w n . F o r static CO2 e x p o s u r e , the n u m b e r o f r e w a r d items eaten, and eating and d w e l l i n g t i m e s s h o w s i m i l a r trends, but eating and d w e l l i n g t i m e s appear to be m o r e sensitive measures, at least at l o w e r concentrations o f CO2. W h i l e eating and d w e l l i n g t i m e s indicate the m a x i m u m t i m e rats are w i l l i n g to tolerate exposure, the n u m b e r o f r e w a r d items eaten p r o v i d e s a measure o f the rats' a c t i v i t y i n the test cage o v e r the entire testing procedure and is affected b y re-entries. F u r t h e r m o r e , rats c a n c o n s u m e the same a m o u n t i n a shorter exposure p e r i o d b y i n c r e a s i n g eating speed. A l t h o u g h rats ate the same a m o u n t w i t h air and 5%> CO2, at 5%> CO2 the m e d i a n t i m e spent eating w a s s l i g h t l y r e d u c e d w i t h o u t an increase i n entries, suggesting that they s i m p l y ate m o r e q u i c k l y . T h e responses o f rats i n the current study m a y not be i n d i c a t i v e o f h o w rats w o u l d r e s p o n d i f e x p o s e d to CO2 for the first t i m e . In order to p e r f o r m w i t h i n - a n i m a l c o m p a r i s o n s it was  necessary  to r e m o v e  the effects o f n o v e l t y  by  familiarizing animals with  all  C0  2  concentrations and d e l i v e r y methods p r i o r to testing. Rats tended to be less tolerant o f static CO2 d u r i n g t r a i n i n g , suggesting that n o v e l t y m a d e exposure m o r e a v e r s i v e . T h u s , b y r e m o v i n g the effects o f n o v e l t y w e have obtained a better i n d i c a t i o n o f a v e r s i o n to the properties o f the gas itself. A n i m a l s were not trained w i t h a r g o n , but it is a n inert gas w i t h no perceptible o d o u r , so a 74  n o v e l t y response to o d o u r w o u l d not be expected. Increases i n i n s p i r e d CO2 and reductions i n i n s p i r e d O2 b o t h cause an i n i t i a l increase i n v e n t i l a t i o n rate and depth ( L u m b , 2 0 0 0 ) , w h i c h c o u l d also contribute to n o v e l t y . H o w e v e r , because h y p e r c a p n i a and h y p o x i a have s i m i l a r effects o n v e n t i l a t i o n , t r a i n i n g w i t h CO2 w o u l d l i k e l y also have b e e n effective for  argon  exposure. O u r results demonstrate that a p p r o a c h - a v o i d a n c e testing c a n p r o v i d e a sensitive a n d objective m e t h o d for e x a m i n i n g rats' a v e r s i o n to gas euthanasia agents. T h e  majority  of  p r e v i o u s studies e x a m i n i n g distress associated w i t h CO2 have r e c o r d e d b e h a v i o u r a l responses d u r i n g p r e - f i l l or g r a d u a l - f i l l euthanasia, and the results have b e e n v a r i a b l e . S o m e studies have reported n o b e h a v i o u r a l evidence o f distress d u r i n g C 0  2  exposure (Hornett & H a y n e s , 1 9 8 4 ;  S m i t h & H a r r a p , 1 9 9 7 ; H a c k b a r t h et a l . , 2 0 0 0 ) . H o w e v e r , others have c o n d u c t e d subjective assessments o f distress ( I w a r s s o n & R e h b i n d e r , 1 9 9 3 ; C o e n e n et a l . , 1995) a n d t a k e n objective b e h a v i o u r a l measures ( B r i t t , 1 9 8 7 ; Chapter 2) and c o n c l u d e d that i n d i c a t i o n s o f distress were present. S o m e v a r i a b i l i t y a m o n g experiments m a y be due to differences i n the w a y b e h a v i o u r a l responses w e r e interpreted. F o r e x a m p l e , B r i t t (1987) reported that Sprague D a w l e y  rats  e x h i b i t e d increased a c t i v i t y and L i s t e r H o o d e d rats e x h i b i t e d decreased a c t i v i t y , yet  both  responses were interpreted as i n d i c a t i v e o f ' d i s c o m f o r t ' . T h e current m e t h o d o l o g y p r o v i d e s a measure o f a v e r s i o n that is less o p e n to subjective interpretation. Preference testing has p r e v i o u s l y  b e e n used to determine whether rats w i l l  exposure to 2 5 . 5 , 3 4 . 9 and 5 0 . 8 % CO2 ( L e a c h et a l . , 2 0 0 2 ) . T h i s p r e v i o u s study  avoid  reported  consistent a v o i d a n c e at a l l three CO2 concentrations, but i m p o s e d n o cost to l e a v i n g the c h a m b e r , m a k i n g it d i f f i c u l t to assess the strength o f a v e r s i o n to CO2. W i t h the  current  a p p r o a c h - a v o i d a n c e d e s i g n , rats h a d to f o r g o a f o o d r e w a r d to a v o i d CO2 exposure. W e c a n therefore c o n c l u d e that the rats' m o t i v a t i o n to a v o i d CO2 at concentrations above 15%> is stronger than their m o t i v a t i o n to o b t a i n a palatable f o o d r e w a r d w h e n f e d ad l i b i t u m . A l t h o u g h 75  the strength o f rats' m o t i v a t i o n for sweet f o o d s w h e n f e d ad l i b i t u m has not s p e c i f i c a l l y been investigated i n the current study, there is e v i d e n c e to suggest that it ranges f r o m moderate to h i g h . In the present study rats w e r e q u i c k to enter the cage f o l l o w i n g l o c k r e m o v a l , and c o n s u m e d the entire f o o d r e w a r d d u r i n g a l l sessions w i t h air. F u r t h e r m o r e , p r e v i o u s studies have s h o w n that m o t i v a t i o n for sucrose w h e n f e d ad l i b i t u m is as m u c h as 5 0 - 7 5 % o f the m o t i v a t i o n for sucrose w h e n f o o d d e p r i v e d . M c G r e g o r et a l . (1999) trained rats to l i c k a sipper tube for access to 8 . 6 % sucrose, and d u r i n g a 4 5 m i n u t e test session w i t h a progressive ratio schedule o f r e w a r d it w a s f o u n d that ad l i b i t u m f e d rats l i c k e d a p p r o x i m a t e l y 1500 t i m e s , w h i l e f o o d d e p r i v e d rats l i c k e d a p p r o x i m a t e l y 2 0 0 0 t i m e s . T h e m a x i m u m n u m b e r o f l i c k s for a s i n g l e r e w a r d w a s a p p r o x i m a t e l y 55 for ad l i b i t u m rats and 7 0 for f o o d - d e p r i v e d rats. In another study rats w e r e trained to bar press for access to sucrose, and it w a s f o u n d that ad l i b i t u m f e d rats bar pressed a p p r o x i m a t e l y 5 5 % as m u c h as f o o d - d e p r i v e d rats d i d for access to 4 % and 16%> sucrose ( C o l l i e r and B o l l e s , 1968). T h e s e results suggest that rats i n this e x p e r i m e n t were w e l l m o t i v a t e d b y the f o o d r e w a r d , and thus indicates that the rats' m o t i v a t i o n to a v o i d exposure to CO2 concentrations o f 1 5 % and greater w a s at least as moderate. T h e strength o f rats' a v e r s i o n to CO2 c o u l d be m o r e accurately assessed i n future studies b y i n c r e a s i n g hunger l e v e l s to ensure that m o t i v a t i o n for the f o o d r e w a r d is h i g h . A p p r o a c h - a v o i d a n c e testing has also been used to e x a m i n e a v e r s i o n to CO2 i n other species. T h e m a j o r i t y o f these studies have e x a m i n e d moderate to h i g h CO2 concentrations because gas s t u n n i n g regulations f o r f a r m a n i m a l s generally require p r e - f i l l exposure (e.g., E u r o p e a n U n i o n , 1993). C o o p e r et a l . (1998) f o u n d that m i n k w i l l a v o i d a c h a m b e r c o n t a i n i n g m o r e than 8 0 % CO2, e v e n w h e n it contains a n o v e l object that they are m o t i v a t e d to o b t a i n . P i g s have b e e n f o u n d to a v o i d a f o o d r e w a r d w h e n it is p a i r e d w i t h 90%  CO2, e v e n f o l l o w i n g f o o d  d e p r i v a t i o n , but w i l l tolerate moderate durations o f exposure to 30%> CO2 (Raj &  Gregory,  1995). P r e v i o u s studies w i t h p o u l t r y have f o u n d that turkeys a n d c h i c k e n s w i l l enter CO2 76  concentrations greater than 60% for a reward o f food or social contact, and w i l l lose consciousness before they are able to exit the chamber (Raj, 1996; Gerritzen et al., 2000; Webster & Fletcher, 2004). This suggests that poultry have a greater tolerance for C 0  2  exposure, but it is not clear whether this interspecific variability demonstrates a difference i n gas perception or in motivation to obtain the reward. The birds were found to exhibit behavioural and physiological signs o f distress during exposure such as hyperventilation, coughing and head shaking, suggesting that the gas was likely detectable and unpleasant. CO2 euthanasia is widely used because it is easy to perform, inexpensive, safe for laboratory workers, and involves little handling and restraint for animals. Another gas euthanasia agent which meets these criteria is argon gas, which causes unconsciousness and death by O2 displacement. In pigs and poultry, unconsciousness and death occur at argon concentrations greater than 90%>, which lowers O2 concentrations below 2% (e.g., Raj, 1999; Raj & Tserveni-Gousi, 2000). During approach-avoidance testing, pigs, turkeys and chickens have been found to enter and remain in lethal concentrations o f argon for a food reward (Raj & Gregory, 1995; Raj, 1996; Webster & Fletcher, 2004). During preference testing, rats and mice have been found to tolerate argon exposure for longer than CO2 exposure, but to exit before loss of consciousness for both gases (Leach et al., 2002). This suggests some level o f aversion with both CO2 and argon. In the current study we found that when the test cage contained 90% argon, some rats refused to enter and others exited immediately. This indicates that rats can detect argon-induced hypoxia and that they find lethal argon concentrations aversive. Argon is an odourless and non-irritating gas, so this aversion is not likely to be due to the properties o f argon. However, low levels o f inspired O2 results in hypoxia, and this could cause a sensation o f dyspnea. The O2 concentration in ambient air is 20.9%, and Moosavi et al. (2003) found that humans report dyspnea at less than 8% O2 when breathing is constrained. This sensation was alleviated with spontaneous breathing, but O2 concentrations less than 1% were not examined.  77  H o w e v e r , h u m a n p i l o t s have b e e n f o u n d to lose consciousness after c a b i n depressurization w i t h o u t apparent efforts at c o r r e c t i o n , i n d i c a t i n g that h u m a n s m a y not experience d y s p n e a w i t h h y p o x i a l e v e l s that are s u f f i c i e n t to cause loss o f consciousness ( C a b l e , 2 0 0 3 ) . T h e d e s i g n o f the current study does not a l l o w for direct c o m p a r i s o n s b e t w e e n responses to CO2 and a r g o n , but it appears that rats are not w i l l i n g to tolerate exposure to either gas for s u f f i c i e n t periods to cause unconsciousness. In h u m a n s , there is a delay to onset o f d y s p n e a after a change i n i n s p i r e d l e v e l s o f CO2 or O2. T h i s d e l a y is due to the t i m e necessary for changes i n b l o o d CO2 and O2 l e v e l s to reach the p e r i p h e r a l and central chemoreceptors, w h i c h is about 5 to 15 s i n h u m a n s ( r e v i e w e d b y C u n n i n g h a m et a l . , 1986), and for h y p o x i a a n d h y p e r c a p n i a to r e a c h l e v e l s that are s u f f i c i e n t to e v o k e d y s p n e a . B a n z e t t (1996) c a l c u l a t e d the h a l f - t i m e for d e v e l o p m e n t o f a stable l e v e l o f d y s p n e a i n h u m a n s to be a p p r o x i m a t e l y 32 s. In the current study, the m e d i a n latency to leave w i t h 2 0 % CO2 and w i t h 9 0 % argon exposure w a s o n l y 5 s and 3 s, r e s p e c t i v e l y , and this response w a s m u c h q u i c k e r than the t i m e t a k e n f o r d y s p n e a to d e v e l o p i n h u m a n s . H o w e v e r , the c i r c u l a t o r y d e l a y is o n l y 2 s i n rats ( L a g n e a u x , 1986), and the d y n a m i c s o f d y s p n e a i n rats is u n k n o w n , so d y s p n e a cannot be r u l e d out as a potential source o f a v e r s i o n d u r i n g b o t h CO2 and a r g o n exposure i n the current study. O u r results suggest that p r e - f i l l and g r a d u a l - f i l l CO2 exposure, and 90%> argon exposure, cause a v e r s i o n i n rats. T h e y also indicate that a p p r o a c h - a v o i d a n c e testing is a sensitive and objective m e t h o d for assessing a v e r s i o n to gas euthanasia m e t h o d s i n rats. Further w o r k is needed to assess h o w a v e r s i o n to CO2 compares w i t h other gas a n d n o n - g a s methods euthanasia, so that the m o s t h u m a n e methods o f euthanasia c a n be i m p l e m e n t e d .  78  of  Table 3.1. M e d i a n  (with 2 5  t h  and 7 5  t h  percentiles) n u m b e r o f r e w a r d items eaten d u r i n g the  entire session and eating and d w e l l i n g t i m e s d u r i n g the first entry w i t h either air o r a r g o n i n the test cage (n = 9 rats).  „ T  ~ y  p  e  0  f  g  W i l c o x o n Signed a  S  R a n k s test  Air  Argon  R e w a r d items eaten (no.)  20(20,20)  0(0,0)  22.5  <0.005  E a t i n g t i m e (s)  261  0(0,0)  22.5  <0.005  D w e l l i n g t i m e (s)  296(296,297)  3 (3,6)  22.5  <0.005  (252,281)  79  250 -i 200 CD 150 E h100 50 -  <> / 4 g>  * - —»  iS 3 H E  2  < 1 —I  1  5  10  1 —  15  20  C a r b o n d i o x i d e c o n c e n t r a t i o n (%)  F i g u r e 3.1.  Median (± interquartile ranges) a) number o f reward items eaten, b) eating time  (filled squares) and dwelling time (open squares) for the first entry, and c) number o f attempted entries into the test cage during sessions with 0, 5, 10, 15 and 20% CO2 (n = 9 rats).  80  3.5 References A n t o n , F., E u c h n e r , I., H a n d w e r k e r , H . O . , 1 9 9 2 . P s y c h o p h y s i c a l e x a m i n a t i o n o f p a i n i n d u c e d b y d e f i n e d CO2 pulses a p p l i e d to the nasal m u c o s a . P a i n 4 9 , 5 3 - 6 0 . A n t o n , F., P e p p e l , P., E u c h n e r , I., H a n d w e r k e r , H . O . , 1 9 9 1 . C o n t r o l l e d n o x i o u s c h e m i c a l s t i m u l a t i o n : responses o f rat t r i g e m i n a l b r a i n s t e m neurones to CO2 pulses a p p l i e d to the nasal m u c o s a . N e u r o s c i . Lett. 1 2 3 , 2 0 8 - 2 1 1 . B a n z e t t , R . B . , 1996. D y n a m i c response characteristics o f C C ^ - i n d u c e s air hunger. R e s p . Physiol. 105, 47-55.  ,  B l a c k s h a w , J . K . , F e n w i c k , D . C . , Beattie, A . W . , A l l a n , D.J., 1988. The behaviour o f chickens, m i c e and rats d u r i n g euthanasia w i t h c h l o r o f o r m , c a r b o n d i o x i d e and ether. L a b . A n i m . 2 2 , 67-75. B r i t t , D . P., 1987. T h e humaneness o f c a r b o n d i o x i d e as a n agent o f euthanasia for laboratory rodents. In: E u t h a n a s i a o f U n w a n t e d , Injured or D i s e a s e d A n i m a l s or f o r E d u c a t i o n a l or S c i e n t i f i c P u r p o s e s , p p . 19-3.1. Potter's B a r : U n i v e r s i t i e s F e d e r a t i o n for A n i m a l W e l f a r e . C a b l e , G . G . , 2 0 0 3 . I n - f l i g h t h y p o x i a incidents i n m i l i t a r y aircraft: causes and i m p l i c a t i o n s for t r a i n i n g . A v i a t . Space E n v i r o n . M e d . 7 4 , 1 6 9 - 1 7 2 . C h a p i n , J . L . , E d g a r , J . L . R . 1 9 6 3 . C o o l i n g o f rats i n c a r b o n d i o x i d e . A m . J . P h y s i o l . 2 0 4 , 7 2 3 726. C h e n , X . , G a l l a r , J . , P o z o , M . A . , B a e z a , M . , B e l m o n t e , C , 1995. C 0  2  s t i m u l a t i o n o f the c o r n e a :  a c o m p a r i s o n b e t w e e n h u m a n sensation and nerve a c t i v i t y i n p o l y m o d a l n o c i c e p t i v e afferents o f the cat. E u r . J . N e u r o s c i . 7, 1 1 5 4 - 1 1 6 3 . C o e n e n , A . M . , D r i n k e n b u r g , W . H . , H o e n d e r k e n , R., v a n L u i j t e l a a r , G . L . , 1 9 9 5 . C a r b o n d i o x i d e euthanasia i n rats: o x y g e n supplementation m i n i m i z e s signs o f agitation a n d a s p h y x i a . L a b . A n i m . 29, 262-268.  81  C o l l i e r , G . , B o l l e s , R., 1 9 6 8 . H u n g e r , thirst, and their interaction as determinants o f sucrose consumption. J. C o m p . Physiol. Psych. 66, 633-641. C o o p e r , J . , M a s o n , G . , R a j , M . , 1 9 9 8 . D e t e r m i n a t i o n o f the a v e r s i o n o f f a r m e d m i n k (Mustela vison) to c a r b o n d i o x i d e . V e t . R e c . 1 4 3 , 3 5 9 - 3 6 1 . C u n n i n g h a m , D . J . C . , R o b b i n s , P. A . , W o l f f , C . B . 1986. Integration o f respiratory response to changes i n a l v e o l a r p a r t i a l pressures o f C 0  2  and O2 and i n arterial p H . In: C h e r n i a k , N . S . ,  W i d d i c o m b e , J . G . (eds), H a n d b o o k o f P h y s i o l o g y , S e c t i o n 3 : T h e R e s p i r a t o r y S y s t e m , V o l u m e II: C o n t r o l o f B r e a t h i n g , Part 2 , A m e r i c a n P h y s i o l o g i c a l S o c i e t y , W a s h i n g t o n , D . C . , pp.475-528 D a n n e m a n , P . J . , S t e i n , S . , W a l s h a w , S . O . , 1997. H u m a n e and p r a c t i c a l i m p l i c a t i o n s o f u s i n g c a r b o n d i o x i d e m i x e d w i t h o x y g e n for anaesthesia or euthanasia o f rats. L a b . A n i m . S c i . 4 7 , 376-85. D r i p p s , R . D . , C o m r o e , J . H . , 1 9 4 7 . T h e respiratory and c i r c u l a t o r y response o f n o r m a l m a n to i n h a l a t i o n o f 7.6 and 10.4 per cent CO2 w i t h a c o m p a r i s o n o f the m a x i m a l v e n t i l a t i o n p r o d u c e d b y severe m u s c u l a r exercise, i n h a l a t i o n o f CO2 a n d m a x i m a l v o l u n t a r y h y p e r v e n t i l a t i o n . A m . J . P h y s i o l . 149, 4 3 - 5 1 . E u r o p e a n U n i o n 1 9 9 3 . C o u n c i l D i r e c t i v e 93/119/EC o f 22 D e c e m b e r 1993 o n the protection o f a n i m a l s at the t i m e o f slaughter or k i l l i n g . O f f i c i a l J o u r n a l L 3 4 0 , 31/12/1993, 0 0 2 1 - 0 0 3 4 . F e n g , Y . , S i m p s o n , T. L., 2 0 0 3 . N o c i c e p t i v e sensation and sensitivity e v o k e d f r o m h u m a n c o r n e a and c o n j u n c t i v a s t i m u l a t e d b y CO2. Invest. O p h t h . V i s . S c i . 4 4 , 5 2 9 - 5 3 2 . G e r r i t z e n , M . A . , L a m b o o i j , E., H i l l e b r a n d , S . J . W . , L a n h a a r , J . A . C . , Pieterse, C , 2 0 0 0 . B e h a v i o r a l responses o f broilers to different gaseous atmospheres. P o u l t . S c i . 7 9 , 9 2 8 - 9 3 3 . H a c k b a r t h , H . , K u p p e r s , N . , B o h n e t , W . , 2 0 0 0 . E u t h a n a s i a o f rats w i t h c a r b o n d i o x i d e — a n i m a l w e l f a r e aspects. L a b . A n i m . 3 4 , 9 1 - 9 6 . H o r n e t t , T . D . , H a y n e s , A . R . , 1984. C o m p a r i s o n o f c a r b o n dioxide/air m i x t u r e and nitrogen/air 82  m i x t u r e for the euthanasia o f rodents. D e s i g n o f a s y s t e m for i n h a l a t i o n euthanasia. A n i m a l Technology 35, 93-99. H e w e t t , T . A . , K o v a c s , M . S . , A r t w o h l , J . E . , Bennett, B . T . ,  1 9 9 3 . A c o m p a r i s o n o f euthanasia  methods i n rats, u s i n g c a r b o n d i o x i d e i n p r e - f i l l e d and f i x e d f l o w rate f i l l e d chambers. L a b . A n i m . Sci. 43, 579-582. I w a r s s o n , K . , R e h b i n d e r , C , 1 9 9 3 . A study o f different euthanasia techniques i n g u i n e a p i g s , rats, a n d m i c e . A n i m a l response and p o s t m o r t e m f i n d i n g s . S c a n . J . L a b . A n i m . S c i . 2 0 , 1 9 1 205. L a g n e a u x , D . , 1986. V e n t i l a t o r y responses o f the rat to m i l d h y p e r c a p n i s t i m u l a t i o n before and after a l m i t r i n e b i s m e s y l a t e . R e s p . P h y s i o l . 6 5 , 3 7 9 - 3 8 8 . L e a c h , M . C , B o w e l l , V . A . , A l l a n , T . F . , M o r t o n , D . B . , 2 0 0 2 . A v e r s i o n to gaseous euthanasia agents i n rats a n d m i c e . C o m p a r a t i v e M e d . 5 2 , 2 4 9 - 2 5 7 . L i o t t i , M . , B r a n n a n , S . , E g a n , G . , Shade, R., M a d d e n , L., A b p l a n a l p , B . , R o b i l l a r d , R., L a n c a s t e r , J . , Z a m a r r i p a , F . E . , F o x , P.T., D e n t o n , D . , 2 0 0 1 . B r a i n responses associated w i t h c o n s c i o u s n e s s o f breathlessness (air hunger). P r o c . N a t . A c a d . S c i . 9 8 , 2 0 3 5 - 2 0 4 0 . L u m b , A . B . , 2000. N u n n ' s A p p l i e d Respiratory Physiology. Butterworth-Heinemann, W o b u r n , M A , pp. 82-106. M c G r e g o r , I.S., S a h a r o v , T., H u n t , G . E . , T o p p l e , A . N . , 1 9 9 9 . B e e r c o n s u m p t i o n i n rats: the i n f l u e n c e o f ethanol content, f o o d d e p r i v a t i o n , and c o c a i n e . A l c o h o l 17, 4 7 - 5 6 . M o o s a v i , S . H . , G o l e s t a n i a n , E., B i n k s , A . P . , L a n s i n g , R . W . , B r o w n , R., B a n z e t t , R . B . , 2 0 0 3 . H y p o x i c a n d h y p e r c a p n i c drives to breathe generate equivalent l e v e l s o f air hunger i n humans. J . A p p l . Physiol. 94, 141-154. P e p p e l , P., A n t o n , F., 1 9 9 3 . R e s p o n s e s o f rat m e d u l l a r y d o r s a l h o r n neurons f o l l o w i n g intranasal n o x i o u s c h e m i c a l s t i m u l a t i o n : effects o f s t i m u l u s intensity, d u r a t i o n , and interstimulus interval. J . Neurophysiol. 70, 2260-2275. 83  R a j , A . B . M . , 1 9 9 6 . A v e r s i v e reactions o f turkeys to argon, c a r b o n d i o x i d e and a m i x t u r e o f c a r b o n d i o x i d e and a r g o n . V e t . R e c . 1 3 8 , 5 9 2 - 5 9 3 . R a j , A . B . M . , 1 9 9 9 . B e h a v i o u r o f pigs e x p o s e d to m i x t u r e s o f gases and the t i m e required to stun and k i l l t h e m : w e l f a r e i m p l i c a t i o n s . V e t . R e c . 144, 1 6 5 - 1 6 8 . R a j , A . B . M . , G r e g o r y , N . G . , 1995. W e l f a r e i m p l i c a t i o n s o f the gas s t u n n i n g o f p i g s 1. D e t e r m i n a t i o n o f a v e r s i o n to i n i t i a l i n h a l a t i o n o f c a r b o n d i o x i d e or argon. A n i m . W e l f a r e 4 , 273-280. R a j , A . B . M . , T s e r v e n i - G o u s i , A . , 2 0 0 0 . S t u n n i n g m e t h o d s for p o u l t r y . W o r l d P o u l t r y S c i . J . 5 6 , 291-304. S m i t h , W . , H a r r a p , S . B . , 1 9 9 7 . B e h a v i o u r a l and c a r d i o v a s c u l a r responses o f rats to euthanasia u s i n g c a r b o n d i o x i d e gas. L a b . A n i m . 3 1 , 3 3 7 - 3 4 6 . T h u r a u f , N . , G u n t h e r , M . , P a u l i , E., K o b a l , G . , 2 0 0 2 . S e n s i t i v i t y o f the negative m u c o s a l p o t e n t i a l to the t r i g e m i n a l target s t i m u l u s CO2. B r a i n R e s . 9 4 2 , 2 7 - 8 6 . W e b s t e r , A . B . , F l e t c h e r , D . L . , 2 0 0 4 . A s s e s s m e n t o f the a v e r s i o n o f hens to different gas atmospheres u s i n g an a p p r o a c h - a v o i d a n c e test. A p p l . A n i m . B e h a v . S c i . 8 8 , 2 7 5 - 2 8 7 .  84  CHAPTER 4: Effect of flow rate on aversion to gradual-fill carbon dioxide euthanasia in rats 4  4.1 Introduction L a b o r a t o r y rats are c o m m o n l y euthanized u s i n g CO2, w h i c h induces u n c o n s c i o u s n e s s f o l l o w e d b y death. A n i m a l s are either p l a c e d into a c h a m b e r that has been p r e - f i l l e d w i t h CO2, or the c h a m b e r c o n t a i n i n g the a n i m a l s is g r a d u a l l y f i l l e d w i t h CO2 u n t i l death is c o n f i r m e d . H o w e v e r , the t e r m ' g r a d u a l ' encompasses a large range o f f l o w rates, and o p t i m a l f l o w rates for m i n i m i z i n g distress have not yet been i d e n t i f i e d . I d e a l l y , a m e t h o d o f euthanasia s h o u l d i n duce death q u i c k l y w i t h o u t c a u s i n g p a i n or distress. CO2 concentrations o f greater than 8 % are k n o w n to cause d y s p n e a , w h i c h is an unpleasant sensation o f breathlessness, i n h u m a n s ( D r i p p s & C o m r o e , 1 9 4 7 ; L i o t t o et a l . , 2 0 0 1 ) , and m a y cause s i m i l a r sensations i n a n i m a l s . CO2 also f o r m s c a r b o n i c a c i d o n the m u c o u s m e m b r a n e s and is k n o w n to cause p a i n i n h u m a n s at concentrations greater than 3 0 to 5 0 % ( A n t o n et a l . , 1 9 9 2 ; C h e n et a l . , 1 9 9 5 ; F e n g &  S i m p s o n , 2 0 0 3 ; T h u r a u f et a l . , 2 0 0 2 ) .  N o c i c e p t o r s i n the nasal m u c o s a and c o r n e a o f rats are also s t i m u l a t e d b y C 0  2  ( P e p p e l and  A n t o n , 1 9 9 3 ; H i r a t a et a l . , 1999), suggesting CO2 l i k e l y also causes p a i n i n rats. D u r i n g g r a d u a l f i l l CO2 euthanasia, faster f l o w rates cause loss o f consciousness and death m o r e q u i c k l y (Hornett & H a y n e s , 1 9 8 4 ; C o e n e n et a l . , 1995). H o w e v e r , w i t h faster f l o w rates a n i m a l s lose c o n s c i o u s n e s s at higher CO2 concentrations ( A m b r o s e et a l . , 2 0 0 0 ) , l i k e l y because loss o f c o n s c i o u s n e s s d u r i n g CO2 exposure is dependent o n p H changes i n the cerebral s p i n a l f l u i d and s l o w f i l l rates a l l o w m o r e t i m e for these p H changes to occur. T h i s exposure to higher CO2 concentrations before loss o f consciousness m a y increase the severity o f any p a i n and d y s p n e a that occur. T h u s , the potential for distress m a y v a r y w i t h f l o w rate; h i g h f l o w rates w i l l expose  A version of this chapter has been accepted for publication. Niel, L . , Stewart, S.A., Weary, D . M . 2006. Effect of flow rate on aversion to gradual-fill carbon dioxide euthanasia in rats. Appl. Anim. Behav. Sci. (accepted).  4  85  a n i m a l s to gas concentrations w i t h a h i g h potential for c a u s i n g distress for a short p e r i o d , w h i l e l o w f l o w rates w i l l cause p r o l o n g e d exposure to gas concentrations w i t h a l o w to moderate potential for c a u s i n g distress. It has b e e n suggested a n e c d o t a l l y that a s l o w l y i n c r e a s i n g CO2 c o n c e n t r a t i o n a l l o w s for gradual onset o f u n c o n s c i o u s n e s s , w i t h o u t the a n i m a l e x p e r i e n c i n g aversive  CO2  concentrations.  The  interaction  between  flow  rate  and  maximum  CO2  c o n c e n t r a t i o n before u n c o n s c i o u s n e s s indicates that this m a y be p l a u s i b l e , s u c h that a range o f s l o w f l o w rates m a y a l l o w for euthanasia w i t h o u t c a u s i n g distress. R a t s ' reactions to g r a d u a l - f i l l CO2 euthanasia have b e e n assessed u s i n g b o t h b e h a v i o u r a l responses to exposure a n d preference testing. W h i l e s o m e studies have reported a l a c k o f b e h a v i o u r a l response to g r a d u a l - f i l l CO2 euthanasia ( H a c k b a r t h et a l . , 2 0 0 0 ; Hornett & H a y n e s , 1 9 8 4 ; S m i t h & H a r r a p , 1997), others have reported b e h a v i o u r a l responses that suggest distress ( B r i t t , 1 9 8 7 ; C o e n e n et a l . , 1 9 9 5 ; Chapter 2). T w o studies have s p e c i f i c a l l y e x a m i n e d the effect o f f l o w rate d u r i n g g r a d u a l - f i l l CO2 exposure i n rats. H o r n e t t and H a y n e s ( 1 9 8 4 ) d i d not find an effect o f f l o w rate o n b e h a v i o u r a l responses, but C o e n e n et a l . (1995) f o u n d that a rate o f 1 2 5 % o f the c h a m b e r v o l u m e per m i n u t e c a u s e d greater g a s p i n g t h a n 14%> o f the c h a m b e r v o l u m e per m i n u t e . T h i s increase i n g a s p i n g suggests a potential for d y s p n e a w i t h the faster f l o w rate. In C h a p t e r 3 w e f o u n d that rats s h o w a v e r s i o n to g r a d u a l - f i l l CO2 exposure at a rate o f 17%> per m i n u t e , but the effects o f f l o w rate were not e x a m i n e d . T h e a i m o f this study w a s to use a p p r o a c h - a v o i d a n c e testing to determine whether a v e r s i o n to g r a d u a l - f i l l euthanasia varies w i t h flow rate.  86  4.2 Materials and Methods 4.2.1 Subjects a n d H o u s i n g T h e subjects were eight 8 - m o n t h o l d , m a l e W i s t a r rats destined f o r euthanasia as surplus stock f r o m the U B C R o d e n t B r e e d i n g U n i t . A n i m a l r o o m s were kept at 21 ± 1 °C under a 1 2 : 1 2 hr l i g h t - d a r k c y c l e , a n d rats w e r e g i v e n a d l i b i t u m access to f o o d ( L a b D i e t 5 0 0 1 , P M I N u t r i t i o n International, R i c h m o n d , U S A ) a n d tap water. A l l testing w a s c o n d u c t e d d u r i n g the light p o r t i o n o f the l i g h t - d a r k c y c l e . R a t s were s i n g l y h o u s e d i n the testing apparatus, c o n s i s t i n g o f t w o transparent cages connected b y a s l o p e d , opaque t u n n e l . T h e top o r ' h o m e ' cage m e a s u r e d 4 8 x 38 x 2 0 c m , a n d c o n t a i n e d f o o d , water, b e d d i n g , an opaque nestbox a n d a N y l a b o n e d o g c h e w . T h e b o t t o m or 'test' cage m e a s u r e d 4 5 x 2 4 x 2 0 c m a n d contained b e d d i n g . T h e h o m e cage w a s 2 7 c m higher than the test cage, a n d the c o n n e c t i n g tunnel w a s m a d e o f b l a c k , r i b b e d , P V C t u b i n g w i t h a diameter o f 10 c m .  4.2.2 T e s t i n g P r o c e d u r e D u r i n g e x p e r i m e n t a l testing, each a n i m a l a n d its testing apparatus w e r e  transferred  i n d i v i d u a l l y to a separate r o o m . A t this t i m e , the w i r e l i d o n the test cage w a s r e p l a c e d w i t h a p l e x i g l a s l i d fitted w i t h a gas inlet i n the center, t w o a i r outlets (1.5 c m i n diameter) p o s i t i o n e d at the e n d closest to the t u n n e l , a n d a gas s a m p l i n g tube inserted at the far e n d o f the test cage. T h e a i r outlets w e r e c o v e r e d w i t h m e s h t o prevent the a n i m a l s f r o m p u s h i n g their noses outside the test cage. A p a r t i t i o n w a s p l a c e d b e h i n d the e x p e r i m e n t a l set-up to c o n c e a l the experimenter d u r i n g testing. B e c a u s e the test cage opened d i r e c t l y into the t u n n e l , the total v o l u m e (24 L ) w a s calculated to i n c l u d e the v o l u m e o f the test cage p l u s the v o l u m e o f the p o r t i o n o f the tunnel that w a s at the same height as the test cage. P r e l i m i n a r y testing w a s c o n d u c t e d to e x a m i n e v a r i a b i l i t y 87  i n CO2 concentrations i n the test cage d u r i n g the f i l l i n g process. CO2 concentrations were m o n i t o r e d at a depth o f h a l f the test cage height at 5 different sites w i t h f l o w rates r a n g i n g f r o m 5 % to 2 8 % o f the test cage v o l u m e per m i n u t e , and were f o u n d to v a r y b y less than 3 ^ 5 % w i t h any g i v e n f l o w rate. F u r t h e r m o r e , there w a s n o o b v i o u s trend for l o w e r CO2 concentrations at e n d o f the test cage closest to the t u n n e l . B e c a u s e CO2 concentrations tend to be greater near the b o t t o m o f the c h a m b e r than at the top ( B r i t t , 1 9 8 7 ; C h a p t e r 2 ) , measurements d u r i n g the e x p e r i m e n t were t a k e n 10 c m above the site o f r e w a r d d e l i v e r y . A i r and CO2 were d e l i v e r e d to the test cage f r o m c o m p r e s s e d gas c y l i n d e r s (Praxair, R i c h m o n d , B . C . ) . T h e treatment gases were passed t h r o u g h a copper c o i l i n a r o o m temperature water bath to regulate the temperature o f the gas before it entered the test cage. F l o w rates o f gases w e r e m e a s u r e d w i t h a v a r i a b l e area f l o w m e t e r ( M o d e l V S B - 6 6 - B V , D w y e r Instruments, Inc.), and m e a s u r e d CO2 f l o w rates were adjusted for density u s i n g a c o r r e c t i o n factor o f 0 . 8 1 2 . R a t s were trained to enter the l o w e r cage for a f o o d r e w a r d , a n d h a d p r e v i o u s l y been tested w i t h exposure to static a n d g r a d u a l l y i n c r e a s i n g concentrations o f CO2 for a separate e x p e r i m e n t . D u r i n g e x p e r i m e n t a l sessions, rats were first l o c k e d i n the top cage f o r 2 m i n . A f t e r the l o c k w a s r e m o v e d the rats w e r e able to enter the l o w e r test cage for a f o o d r e w a r d o f 2 0 H o n e y N u t C h e e r i o s ™ ( G e n e r a l M i l l s , Inc., M i n n e s o t a ) . A s s o o n as the rat entered the test cage and started eating the C h e e r i o s ™ , either air or CO2 flow w a s initiated at a p r e - d e t e r m i n e d rate. R a t s c o u l d r e m a i n i n the test cage f o r a m a x i m u m o f 3 0 0 s f r o m the t i m e that gas flow b e g a n , after w h i c h the test session w a s ended. If the rat entered the h o m e cage d u r i n g this p e r i o d the test session w a s stopped. A t the end o f the session, the r e m a i n i n g r e w a r d items were r e m o v e d a n d c o u n t e d , and the rat w a s returned to the h o l d i n g r o o m . T h e testing apparatus a n d O2 meter readout were v i d e o r e c o r d e d d u r i n g testing. W e also r e c o r d e d the total n u m b e r o f r e w a r d items eaten over the entire test session as w e l l as the  88  latency to stop eating a n d the latency to leave the test cage after gas f l o w h a d b e g u n . G a s concentrations i n the test cage were m o n i t o r e d d u r i n g the e x p e r i m e n t v i a the gas s a m p l i n g tube u s i n g a M o c o n L F 7 0 0 D O2 analyzer. T h e O2 c o n c e n t r a t i o n w a s r e c o r d e d a n d used to calculate C0  2  concentrations at e a c h o f these t i m e s (t = x ) w i t h the f o r m u l a : C02(t= ) = 100 - ( 1 0 0 * ([O2 X  (t = x ) ] / [ O ( , = 2  0)])).  R a t s w e r e tested i n t w o replicates o f eight test sessions, a n d i n e a c h replicate rats w e r e tested o n f i v e days w i t h C 0  a n d o n three c o n t r o l days w i t h air. F o r b o t h replicates, rats were  2  tested w i t h f i v e different C 0  2  f l o w rates: 3 , 7 , 14, 2 0 , a n d 2 7 % o f the test cage v o l u m e per  m i n u t e . In the first r e p l i c a t e , a f l o w rate o f 2 1 % p e r m i n u t e w a s used f o r a l l three test sessions w i t h air. In the s e c o n d replicate, f l o w rates o f 4 , 17 a n d 3 3 % per m i n u t e were used f o r the three test sessions w i t h air. Treatment order f o r CO2 a n d air w a s b a l a n c e d across rats a n d days b y a n 8 x 8 L a t i n square. O n l y f l o w rates less than 30%> p e r m i n u t e were e x a m i n e d because the results o f Ambrose  et a l . (2000)  suggest  that faster f l o w  rates result  i n potentially  p a i n f u l CO2  concentrations before a n i m a l s lose c o n s c i o u s n e s s .  4.2.3 Statistical Analysis D a t a w e r e averaged w i t h i n rat a n d CO2 f l o w rate f o r the t w o replicates, r e s u l t i n g i n 4 0 observations f o r the a n a l y s i s o f CO2 f l o w rate (8 rats a n d 5 f l o w rates). F o r the a n a l y s i s o f air f l o w rate, the three f l o w rates f r o m the s e c o n d replicate were e x a m i n e d , r e s u l t i n g i n 2 4 observations (8 rats a n d 3 f l o w rates). D e p e n d e n t v a r i a b l e s were a n a l y z e d u s i n g a m i x e d m o d e l ( S A S v 9 . 1 ) that i n c l u d e d rat (7 d.f.) as a r a n d o m effect, a n d tested f o r linear a n d quadratic effects o f f l o w rate (1 d.f. f o r each) against a n error t e r m w i t h 3 0 d.f. f o r the test o f CO2 f l o w rate a n d 14 d.f. f o r the test o f air f l o w rate. L a t e n c y to leave the test cage w a s not tested i n the air f l o w a n a l y s i s because a l l a n i m a l s r e m a i n e d i n the test cage f o r the entire testing p e r i o d .  89  4.3 Results D u r i n g test sessions w i t h air, rats ate o n average ( ± S E ) 19.3 ± 0.3 r e w a r d items out o f 2 0 , and f i n i s h e d eating 2 7 0 ± 6 s after entering the test cage. A l l rats r e m a i n e d i n the test cage for the entire 3 0 0 s testing p e r i o d for a l l air sessions. C h a n g e s i n air f l o w rate d i d not affect the latency to stop eating (linear: F\,u  -  1.74, P > 0 . 1 ; quadratic: F i j 4 = 0 . 1 9 , P > 0.1) or the  n u m b e r o f r e w a r d items eaten (linear: F i j 4 = 0 . 2 9 , P > 0 . 1 ; quadratic: F i j 4 = 0 . 0 7 , P > 0.1). In contrast, the n u m b e r o f r e w a r d items eaten, the latency to stop eating, a n d the latency to leave the test cage decreased w i t h i n c r e a s i n g CO2 f l o w rates ( F i g . 4.1 a, b). B o t h the linear a n d c u r v i l i n e a r effects were s i g n i f i c a n t for the n u m b e r o f r e w a r d items eaten (linear: Fi^o 6 7 . 2 1 , P < 0 . 0 0 1 ; quadratic: F i  > 3 0  = 5 . 0 2 , P < 0 . 0 5 ) , the latency to stop eating (linear: F i ,  =  =  3 0  1 2 8 . 3 6 , P < 0 . 0 0 1 ; quadratic: F o = 1 0 . 9 1 , P < 0 . 0 1 ) , and the latency to leave the test cage U  (linear: F i  > 3 0  = 171.24, P < 0 . 0 0 1 ; quadratic: F  = 11-84, P < 0.01).  h30  T h e rats d i d not r e m a i n i n the test cage for l o n g e n o u g h to lose consciousness at any o f the f l o w rates, but there w a s a c u r v i l i n e a r r e l a t i o n s h i p b e t w e e n CO2 f l o w rate and the CO2 c o n c e n t r a t i o n at the t i m e rats stopped eating ( F i o >3  =  5.65 , P < 0.05) and left the test cage ( F i , o 3  = 9 . 0 2 , P < 0.01). R a t s stopped eating and left the test cage at l o w e r CO2 concentrations w i t h the l o w e s t a n d highest f l o w rates ( F i g . 4.1 c). R a t s left the test cage at the highest CO2 concentration ( 1 5 . 9 % CO2) w h e n tested at the 14%> per m i n u t e f l o w rate. H o w e v e r , the CO2 concentration w h e n rats left the test cage v a r i e d c o n s i d e r a b l y across rats. W h e n averaged w i t h i n rat across a l l days for a l l f l o w rates, the average CO2 concentration w h e n rats left the test cage ranged f r o m 1 1 . 1 % to 18.6%). T h e m a x i m u m and m i n i m u m CO2 concentrations tolerated before rats left the test cage ranged f r o m 4 . 8 % to 25.3%>. V a r i a b i l i t y w a s also o b s e r v e d for i n d i v i d u a l rats; for e x a m p l e , one rat left the test cage at 4 . 8 % CO2 o n one day and at 21.5%) CO2 o n a different day.  90  4.4 Discussion It has b e e n suggested a n e c d o t a l l y that s l o w CO2  f i l l rates c a n result i n loss  of  consciousness i n rats before aversive CO2 concentrations o c c u r . H o w e v e r , rats i n the current study left the test cage before l o s i n g c o n s c i o u s n e s s for a l l test sessions w i t h CO2. T h i s result demonstrates that rats are averse to g r a d u a l - f i l l CO2 exposure w i t h flow rates r a n g i n g f r o m 3 % to 2 7 % o f the test cage v o l u m e per m i n u t e . F l o w rate h a d no effect o n any variables d u r i n g test sessions w i t h air, i n d i c a t i n g that it w a s CO2 exposure that resulted i n a v e r s i o n rather than sound or air currents associated w i t h changes i n gas flow d y n a m i c s . In the current study, the latency to leave the test cage decreased w i t h i n c r e a s i n g  flow  rate, s u c h that o n average rats left the test cage w h e n CO2 concentrations were between 1 3 . 0 % a n d 1 5 . 9 % . In a p r e v i o u s study e x a m i n i n g a v e r s i o n to CO2, rats s h o w e d a v e r s i o n to static CO2 at concentrations o f 1 5 % , a n d to a g r a d u a l l y i n c r e a s i n g c o n c e n t r a t i o n o f CO2 at a p p r o x i m a t e l y 18%> (Chapter 3). T h e s e results indicate that there is a t h r e s h o l d CO2 c o n c e n t r a t i o n that rats find a v e r s i v e , a n d that it is r e l a t i v e l y consistent regardless o f flow rate. T h i s concentration o f CO2 is u n l i k e l y to cause p a i n i n rats. T h e m a j o r i t y o f receptors i n the nasal m u c o s a r e s p o n d to CO2 concentrations between 37 and 5 0 % CO2  (Anton  et a l . , 1 9 9 1 ; P e p p e l &  Anton,  1993).  F u r t h e r m o r e , p a i n f u l s t i m u l a t i o n o f the nasal m u c o s a is k n o w n to e l i c i t apnea and b r a d y c a r d i a , a n d this response is not observed i n rats at CO2 concentrations r a n g i n g f r o m 10%> to 5 0 % ( Y a v a r i et a l . , 1996). H o w e v e r , CO2 concentrations as l o w as 8 % have b e e n associated w i t h a sensation o f d y s p n e a , or breathlessness, i n h u m a n s ( D r i p p s & C o m r o e , 1 9 4 7 ; L i o t t i et a l . , 2 0 0 1 ) , a n d this sensation m a y o c c u r i n rats. W e therefore suggest that d y s p n e a is m o r e l i k e l y to be the cause o f a v e r s i o n i n the current study. T h e CO2 concentration at l e a v i n g t i m e v a r i e d w i t h flow rate, w i t h rats tolerating s l i g h t l y higher CO2 concentrations at intermediate flow rates. A t l o w flow rates, rats are l i k e l y l e a v i n g at  91  l o w e r CO2 concentrations because the extended p e r i o d o f exposure to l o w e r concentrations reduces their o v e r a l l tolerance for CO2. Sensations o f d y s p n e a due to h y p e r c a p n i a are l i k e l y m e d i a t e d b y central and p e r i p h e r a l chemoreceptors ( A m e r i c a n T h o r a c i c S o c i e t y , 1999), w h i c h are sensitive to reductions i n the p H o f b l o o d and cerebral s p i n a l f l u i d . E x t e n d e d  exposure  w o u l d a l l o w greater t i m e for these adjustments, s u c h that the m a x i m u m tolerance is reached at a lower  concentration. R e d u c e d tolerance at h i g h f l o w  rates indicates that CO2  detection  m e c h a n i s m s m i g h t be sensitive to not o n l y absolute CO2 c o n c e n t r a t i o n , but also to the rate at w h i c h CO2 is i n c r e a s i n g . O n l y f l o w rates less than 3 0 % per m i n u t e were e x a m i n e d i n the current study because the results o f A m b r o s e et a l . (2000) suggest that h i g h f l o w rates result i n CO2 concentrations that are s u f f i c i e n t to cause p a i n before u n c o n s c i o u s n e s s i n m i c e . A m b r o s e et a l . (2000) f o u n d that a f l o w rate o f 6 0 % o f the c h a m b e r v o l u m e per m i n u t e resulted i n CO2 concentrations above 50%) i n the 10 s or so before m i c e lost c o n s c i o u s n e s s , w h i l e at 30%  per m i n u t e m i c e b e c a m e  u n c o n s c i o u s at CO2 concentrations under 50%>. T h u s , a l t h o u g h faster f l o w rates result i n a shorter d u r a t i o n o f exposure before loss o f c o n s c i o u s n e s s , s l o w e r f l o w rates m a y prevent exposure to CO2 l e v e l s that are s u f f i c i e n t to cause p a i n . W h i l e f l o w rates greater than 30%) per m i n u t e were not e x a m i n e d , the CO2 concentration at w h i c h rats left the test cage v a r i e d i n a p a r a b o l i c m a n n e r , suggesting that rats w o u l d leave at l o w e r concentrations w i t h - f l o w rates above those tested i n the current study. T h e a p p r o a c h - a v o i d a n c e test used i n the current study indicates that the m a x i m u m CO2 c o n c e n t r a t i o n tolerated varies w i t h f l o w rate, but this study p r o v i d e s little i n f o r m a t i o n o n the l e v e l o f distress that rats w o u l d have e x p e r i e n c e d had they b e e n f o r c e d to r e m a i n i n the test cage u n t i l death. T h i s distress w o u l d be dependent b o t h o n the d u r a t i o n o f the p e r i o d between onset o f a v e r s i o n and u n c o n s c i o u s n e s s , and the strength o f a v e r s i o n to the CO2  concentrations  o c c u r r i n g d u r i n g this p e r i o d . P r e v i o u s studies have attempted to e x a m i n e b e h a v i o u r a l responses 92  d u r i n g the entire euthanasia procedure as a means o f assessing distress. W h i l e s o m e studies have f o u n d e v i d e n c e to suggest a distress response ( B r i t t , 1 9 8 7 ; C o e n e n et a l . , 1 9 9 5 ; Chapter 2 ) , others have reported no effect ( H a c k b a r t h et a l . , 2 0 0 0 ; H o r n e t t & H a y n e s , 1 9 8 4 ; S m i t h & H a r r a p , 1997). S o m e o f these studies have s p e c i f i c a l l y c o m p a r e d b e h a v i o u r a l responses d u r i n g g r a d u a l - f i l l CO2 exposure at different flow rates. H o r n e t t and H a y n e s (1984) e x a m i n e d  flow  rates r a n g i n g f r o m 6 to 4 0 % per m i n u t e w i t h rats and w h i l e adverse reactions were not reported f o r any  flow  rate, a rate o f 1 9 . 5 % per m i n u t e w a s r e c o m m e n d e d based o n a  subjective  assessment o f the procedure and t i m e to unconsciousness and death. T i m e to u n c o n s c i o u s n e s s w a s a p p r o x i m a t e l y 4 m i n at 6 % per m i n u t e , but w a s r e d u c e d to a p p r o x i m a t e l y 2 m i n for  flow  rates o f 13 to 40%» per m i n u t e . C o e n e n et a l . (1995) c o m p a r e d flow rates o f 1 4 % and 1 2 5 % per m i n u t e and f o u n d that b o t h treatments resulted a s i m i l a r p e r i o d o f ' e x c i t a t i o n and a g i t a t i o n ' , but that g a s p i n g w a s s l i g h t l y h i g h e r i n the fast fill g r o u p , suggesting i n c r e a s e d d y s p n e a . H o w e v e r , the t i m e to r e c u m b e n c y , aberrant E E G and a b n o r m a l E C G were s i g n i f i c a n t l y longer i n the 1 4 % per minute group. In c o n c l u s i o n , rats s h o w a v o i d a n c e d u r i n g exposure to g r a d u a l - f i l l CO2 exposure w i t h flow  rates r a n g i n g f r o m 3 % to 2 7 % per m i n u t e . T h e CO2 c o n c e n t r a t i o n at the t i m e rats left the  test cage v a r i e d w i t h flow rate, i n d i c a t i n g that a flow rate o f 1 4 % per m i n u t e is o p t i m a l i n terms o f i n i t i a l a v e r s i o n . H o w e v e r , f o r c e d exposure to CO2 b e y o n d this i n i t i a l a v e r s i o n is l i k e l y to result i n distress w i t h a l l flow rates, therefore further research is needed to d e v e l o p m e t h o d s o f euthanasia for laboratory rats.  93  better  a)  ci  8 -.  ten  6 -  ms  c  4-  CO CD  CD ~o 2CD $ CD  0-  10  15  20  25  30  25  30  25  30  Flow rate (%/min) b)  100 „—  -i  80 -  s  (/}  >. 60 o  c  a> 40 ro _i 20 010  15  20  Flow rate (%/min) c)  20 - i ^  16 CD  "O X  12 -  C  8-  o T3 o  ro 4 -  O  010  15  20  Flow rate (%/min) F i g u r e 4 . 1 . L e a s t squares m e a n ( ± S E M ) a) n u m b e r o f r e w a r d items eaten, b) latency to stop eating (open) and to leave the test cage ( f i l l e d ) , and c) CO2 c o n c e n t r a t i o n at the t i m e w h e n rats stopped eating (open) a n d left the test cage ( f i l l e d ) d u r i n g test sessions w i t h CO2 f l o w rates o f 3 , 7, 14, 2 0 a n d 2 7 % o f the test cage v o l u m e per m i n u t e (n = 8 rats).  94  4.5 References A m b r o s e , N . , W a d h a m , J . , M o r t o n , D . , 2 0 0 0 . R e f i n e m e n t i n E u t h a n a s i a . In: B a l l s , M . , v a n Z e l l e r , A . M . , H a i d e r , M . E . ( E d s ) , Progress i n the R e d u c t i o n , R e f i n e m e n t a n d R e p l a c e m e n t of A n i m a l Experimentation, Elsevier Science, Amsterdam, pp.1159-1169. American Thoracic  Society.  1 9 9 9 . D y s p n e a : m e c h a n i s m s , assessment, and m a n a g e m e n t ; a  consensus statement. A m . J . R e s p i r . C r i t . C a r e M e d . 1 5 9 , 3 2 1 - 3 4 0 A n t o n , F., E u c h n e r , I., H a n d w e r k e r , H . O . ,  1992. Psychophysical examination o f pain induced  b y d e f i n e d CO2 pulses a p p l i e d to the nasal m u c o s a . P a i n 4 9 , 5 3 - 6 0 . A n t o n , F.,  P e p p e l , P., E u c h n e r , I.,  Handwerker,  H.O.,  1991. Controlled noxious chemical  s t i m u l a t i o n : responses o f rat t r i g e m i n a l b r a i n s t e m neurones to CO2 pulses a p p l i e d to the nasal m u c o s a . N e u r o s c i . Lett. 1 2 3 , 2 0 8 - 2 1 1 . B r i t t , D . P . , 1 9 8 7 . T h e humaneness o f c a r b o n d i o x i d e as an agent o f euthanasia for laboratory rodents. In: E u t h a n a s i a o f U n w a n t e d , Injured or D i s e a s e d A n i m a l s or for E d u c a t i o n a l or S c i e n t i f i c P u r p o s e s , p p . 1 9 - 3 1 . Potter's B a r : U n i v e r s i t i e s F e d e r a t i o n for A n i m a l W e l f a r e . C h e n , X . , G a l l a r , J . , P o z o , M . A . , B a e z a , M . , B e l m o n t e , C , 1 9 9 5 . CO2 s t i m u l a t i o n o f the c o r n e a : a comparison between  h u m a n sensation and nerve a c t i v i t y  in polymodal  nociceptive  afferents o f the cat. E u r . J . N e u r o s c i . 7, 1 1 5 4 - 1 1 6 3 . C o e n e n , A . M . , D r i n k e n b u r g , W . H . , H o e n d e r k e n , R., v a n L u i j t e l a a r , G . L . , 1995. C a r b o n d i o x i d e euthanasia i n rats: o x y g e n supplementation m i n i m i z e s signs o f agitation and a s p h y x i a . L a b . A n i m . 29, 262-268. D r i p p s , R . D . , C o m r o e , J . H . , 1947. T h e respiratory and c i r c u l a t o r y response o f n o r m a l m a n to i n h a l a t i o n o f 7.6 and 10.4 per cent CO2 w i t h a c o m p a r i s o n o f the m a x i m a l v e n t i l a t i o n produced  by  severe  muscular  exercise,  inhalation  hyperventilation. A m . J. P h y s i o l . 149, 4 3 - 5 1 .  95  of  CO2  and  maximal  voluntary  F e n g , Y . , S i m p s o n , T. L., 2 0 0 3 . N o c i c e p t i v e sensation a n d sensitivity e v o k e d f r o m h u m a n c o r n e a and c o n j u n c t i v a stimulated b y CO2. Invest. O p h t h . V i s . S c i . 4 4 , 5 2 9 - 5 3 2 . H a c k b a r t h , H . , K u p p e r s , N . , B o h n e t , W . , 2 0 0 0 . E u t h a n a s i a o f rats w i t h c a r b o n d i o x i d e — a n i m a l w e l f a r e aspects. L a b . A n i m . 3 4 , 9 1 - 9 6 . H i r a t a , H . , H u , J . W . , Bereiter, D . A . ,  1999. R e s p o n s e s o f m e d u l l a r y dorsal h o r n neurons to  c o r n e a l s t i m u l a t i o n b y CO2 pulses i n the rat. J . N e u r o p h y s i o l . 8 2 , 2 0 9 2 - 2 1 0 7 . H o r n e t t , T . D . , H a y n e s , A . R . , 1984. C o m p a r i s o n o f c a r b o n dioxide/air m i x t u r e and nitrogen/air m i x t u r e for the euthanasia o f rodents. D e s i g n o f a system for i n h a l a t i o n euthanasia. A n i m a l Technology 35, 93-99. L i o t t i , M . , B r a n n a n , S . , E g a n , G . , S h a d e , R.,  M a d d e n , L.,  Abplanalp, B., Robillard,  R.,  L a n c a s t e r , J . , Z a m a r r i p a , F . E . , F o x , P.T., D e n t o n , D . , 2 0 0 1 . B r a i n responses associated w i t h c o n s c i o u s n e s s o f breathlessness (air hunger). P r o c . N a t . A c a d . S c i . 9 8 , 2 0 3 5 - 2 0 4 0 . P e p p e l , P., A n t o n , F., 1 9 9 3 . R e s p o n s e s o f rat m e d u l l a r y dorsal h o r n neurons f o l l o w i n g intranasal n o x i o u s c h e m i c a l s t i m u l a t i o n : effects o f s t i m u l u s intensity, d u r a t i o n , and interstimulus interval. J. Neurophysiol. 70, 2260-2275. S m i t h , W . , H a r r a p , S . B . , 1 9 9 7 . B e h a v i o u r a l and c a r d i o v a s c u l a r responses o f rats to euthanasia u s i n g c a r b o n d i o x i d e gas. L a b . A n i m . 3 1 , 3 3 7 - 3 4 6 . T h u r a u f , N . , G u n t h e r , M . , P a u l i , E., K o b a l , G . , 2 0 0 2 . S e n s i t i v i t y o f the negative m u c o s a l potential to the t r i g e m i n a l target s t i m u l u s CO2. B r a i n R e s . 9 4 2 , 2 7 - 8 6 . Yavari,  P., M c C u l l o c h , P . F . ,  Panneton, W . M . ,  1996. T r i g e m i n a l l y - m e d i a t e d  alteration  of  cardiorespiratory r h y t h m s d u r i n g nasal a p p l i c a t i o n o f c a r b o n d i o x i d e i n the rat. J . A u t o n . N e r v . Syst. 6 1 , 1 9 5 - 2 0 0 .  96  CHAPTER 5: Effects of novelty on rat responses to C 0 exposure  5  2  5.1 Introduction C a r b o n d i o x i d e is w i d e l y  used for k i l l i n g laboratory rodents, but recent  evidence  suggests that exposure to CO2 m a y cause distress and a v e r s i o n before loss o f consciousness i n rats. E x p o s u r e o f rats to a g r a d u a l l y i n c r e a s i n g concentration o f CO2 has been s h o w n to result i n ^ escape b e h a v i o u r s , s u c h as p u s h i n g and s c r a t c h i n g at the c h a m b e r l i d , and increased e x p l o r a t i o n ( C h a p t e r 2). F u r t h e r m o r e , L e a c h et a l . (2002) f o u n d that rats a v o i d CO2 concentrations o f 2 5 . 5 % and greater, a n d , as d e s c r i b e d i n Chapters 3 a n d 4, rats w i l l f o r g o a palatable f o o d r e w a r d i n order to a v o i d CO2 concentrations o f 15%> and greater. B o t h d y s p n e a (an unpleasant sensation o f breathlessness) a n d p a i n have b e e n suggested as potential causes o f distress and a v e r s i o n d u r i n g CO2 euthanasia. CO2 concentrations  of  greater than 30%> are necessary to cause loss o f posture i n rats ( C h a p t e r 2 ; S m i t h and H a r r a p , 1997), and loss o f posture indicates the a p p r o x i m a t e onset o f u n c o n s c i o u s n e s s (e.g. C o e n e n et a l . , 1995). CO2 f o r m s c a r b o n i c a c i d w h e n it c o m e s into contact w i t h m o i s t u r e at the m u c o s a l m e m b r a n e s , a n d starts to cause p a i n i n h u m a n s at concentrations o f 3 0 to 5 0 % ( A n t o n et a l . , 1 9 9 2 ; C h e n et a l . , 1 9 9 5 ; F e n g & S i m p s o n , 2 0 0 3 ; T h u r a u f et a l . , 2 0 0 2 ) . R a t s have n o c i c e p t o r s i n the nasal m u c o s a that r e s p o n d to CO2 at s i m i l a r concentrations ( A n t o n et a l . , 1 9 9 1 ; P e p p e l and A n t o n , 1993), and so rats m a y experience p a i n at moderate CO2 concentrations. CO2 also causes d y s p n e a i n h u m a n s at concentrations o f o n l y 8%> ( D r i p p s & C o m r o e , 1 9 4 7 ; L i o t t i et a l . , 2 0 0 1 ) . In p r e v i o u s studies o n CO2 euthanasia, rats have s h o w n b e h a v i o u r a l signs o f distress and a v e r s i o n d u r i n g exposure to r e l a t i v e l y l o w concentrations o f CO2 (Chapters 2 , 3 and 4). T h e s e  A version of this chapter has been submitted for publication. Niel, L., Weary, D.M. 2006. Effects of novelty on rat responses to C 0 exposure. Appl. Anim. Behav. Sci. (submitted). 5  2  97  responses o c c u r r e d at CO2 concentrations that were l o w e r than n o c i c e p t i v e thresholds, therefore d y s p n e a is a m o r e l i k e l y cause o f distress and a v e r s i o n than p a i n . H o w e v e r , another potential source o f distress d u r i n g CO2 exposure is n o v e l t y .  Novelty  has been suggested to i n d u c e a n a p p r o a c h - a v o i d a n c e c o n f l i c t i n rats, r e s u l t i n g f r o m  an  interaction b e t w e e n e x p l o r a t o r y m o t i v a t i o n and fear ( M o n t g o m e r y , 1955). W a l l a c e and R o s e n (2000) demonstrated that exposure o f rats to n o v e l o d o u r s , such as b u t y r i c a c i d ( s i m i l a r to r a n c i d butter) and i s o a m y l acetate ( s i m i l a r to banana), causes a v o i d a n c e , reduces g r o o m i n g t i m e a n d increases f r e e z i n g t i m e , a n d these responses suggest that n o v e l odours c a n e l i c i t fear. O d o u r p e r c e p t i o n occurs as a result o f s t i m u l a t i o n o f b o t h o l f a c t o r y and t r i g e m i n a l neurons, w i t h the latter c o n t r i b u t i n g m a i n l y to p u n g e n c y ( C a i n a n d M u r p h y , 1980). CO2 is thought to stimulate m a i n l y t r i g e m i n a l neurons i n the nasal m u c o s a , but because o d o u r p e r c e p t i o n occurs as a result o f both o l f a c t o r y a n d t r i g e m i n a l input, h u m a n s perceive an o d o u r q u a l i t y w h e n a s k e d to describe sensations o c c u r r i n g w i t h CO2 i n h a l a t i o n ( C a i n and M u r p h y , 1980). R a t s c a n detect CO2 at concentrations b e t w e e n 0.04 and 1 . 7 % ( Y o u n g e n t o b , 1991), w h i c h is b e l o w the levels required to stimulate t r i g e m i n a l neurons i n rat nasal m u c o s a ( P e p p e l and A n t o n , 1993). T h e exact q u a l i t y o f CO2 that rats are r e s p o n d i n g to is u n k n o w n , but they have a sensitive o l f a c t o r y system and m a y h a v e a n e n h a n c e d a b i l i t y to detect the o d o u r q u a l i t y o f CO2. In order to test whether distress and a v e r s i o n associated w i t h CO2 exposure is due i n part to n o v e l t y , w e used t w o different e x p e r i m e n t a l approaches. F o r the first set o f e x p e r i m e n t s , w e used an a p p r o a c h - a v o i d a n c e test to e x a m i n e gas a v e r s i o n i n rats, by p a i r i n g gas exposure w i t h a f o o d r e w a r d . In E x p e r i m e n t 1 w e e x a m i n e d rat a v e r s i o n to g r a d u a l - f i l l CO2 exposure. Rats were tested w i t h  repeated  CO2  exposure  to  document  whether  their  responses  would  show  habituation. In E x p e r i m e n t 2 w e used the same m e t h o d o l o g y to e x a m i n e h o w a n o v e l odour affects rat p e r f o r m a n c e o n the a p p r o a c h - a v o i d a n c e test. W e u s e d p e p p e r m i n t as the n o v e l o d o u r , and this s t i m u l u s w a s not expected to produce p a i n or respiratory 98  stimulation. For  both  experiments, we compared rat responses to the first exposure and subsequent exposures to determine whether each condition was aversive and whether novelty was a source of aversion, using the following logic. We reasoned that: 1. a lack of aversion would be indicated by similar eating and dwelling times with air and the treatment gas, 2. aversion due to novelty would result in animals eating less and leaving earlier on the initial exposure, but that this reaction would decrease on subsequent exposures, and 3. aversion without an effect of novelty would result in the animals eating less and leaving earlier on all exposures. For the second experimental approach, we examined the behavioural responses of rats during exposure to a gradually increasing concentration of either CO2 or peppermint odour (Experiment 3). We predicted that behaviours that were due to novelty would be present with both treatments, but that those occurring as a result of other factors, such as pain and dyspnea, would occur only in the CO2 treatment group.  5.2 Materials and Methods 5.2.1 S u b j e c t s , H o u s i n g , a n d E q u i p m e n t  Rats were obtained as surplus stock (i.e. animals already slated for euthanasia) from the U B C Rodent Breeding Unit, and housed at 21°C under a 12:12-hr light-dark cycle with ad libitum access to food (Lab Diet 5001, PMI Nutrition International, Indiana, U S A ) and tap water. A l l testing was conducted during the light portion of the light-dark cycle. CO2 and air were delivered from compressed gas cylinders (Praxair, Richmond, B.C.). For some treatments, air was scented with peppermint odour by routing the air flow through a 266 mL chamber containing three cotton balls soaked in 2 mL of peppermint extract (Canada Safeway Ltd., Calgary). The treatment gases were passed through a copper coil in a room temperature water bath to regulate the temperature of the gas before it entered the chamber.  99  F l o w rates o f the gases were m e a s u r e d b y a v a r i a b l e area f l o w m e t e r ( M o d e l V S B - 6 6 - B V , D w y e r Instruments, Inc., M i c h i g a n ) , a n d m e a s u r e d CO2 f l o w rates were adjusted f o r density w i t h a c o r r e c t i o n factor o f 0 . 8 1 2 . G a s concentrations i n the c h a m b e r w e r e m o n i t o r e d d u r i n g the e x p e r i m e n t v i a a gas s a m p l i n g tube u s i n g a M o c o n L F 7 0 0 D O2 a n a l y z e r , a n d the f o l l o w i n g f o r m u l a w a s used to calculate the concentration o f CO2 at s p e c i f i c t i m e points (t = x ) d u r i n g the f i l l i n g process: [ C 0 ( , = ] = 1 0 0 - ( 1 0 0 * ( [ 0 2 ( t = x)]/ [O2 (,-<»])). 2  s)  5.2.2 Experiments 1 & 2: Approach-avoidance Testing W e used a n a p p r o a c h - a v o i d a n c e test to e x a m i n e rats' a v o i d a n c e o f a g r a d u a l a d d i t i o n o f either CO2 ( E x p e r i m e n t 1) or air w i t h p e p p e r m i n t odour ( E x p e r i m e n t 2 ) . T h e p e p p e r m i n t w a s not expected to cause p a i n or respiratory s t i m u l a t i o n . R a t s were s i n g l y h o u s e d i n the testing apparatus c o n s i s t i n g o f t w o transparent cages c o n n e c t e d b y a n opaque t u n n e l m a d e o f b l a c k , r i b b e d , P V C t u b i n g w i t h a diameter o f 10 c m a n d s l o p e d so that one cage w a s 2 7 c m higher than the other. T h e ' h o m e ' cage m e a s u r e d 4 8 x 38 x 2 0 c m , a n d c o n t a i n e d f o o d , water, b e d d i n g , a n opaque nestbox a n d a N y l a b o n e d o g c h e w . T h e secondary cage m e a s u r e d 4 5 x 2 4 x 2 0 c m a n d contained b e d d i n g . T h e l o w e r cage w a s a l w a y s used f o r testing because the test gases were denser than air. D u r i n g p r e l i m i n a r y testing o f the e x p e r i m e n t a l apparatus w e d e t e r m i n e d that the test gases r e m a i n e d i n the l o w e r cage a n d the l o w e r p o r t i o n o f the t u n n e l . F o r E x p e r i m e n t 1, h a l f o f the rats were tested i n the h o m e cage a n d the other h a l f w e r e tested i n the secondary cage f o r the purposes o f a separate experiment ( C h a p t e r 3). F o r E x p e r i m e n t 2 , a l l rats were tested i n the secondary cage. D u r i n g e x p e r i m e n t a l testing, each a n i m a l a n d its testing apparatus w e r e  transferred  i n d i v i d u a l l y to a test r o o m . A t this t i m e , the nest b o x w a s r e m o v e d f r o m the h o m e cage a n d the w i r e l i d o n the test cage w a s replaced w i t h a p l e x i g l a s l i d that featured t w o air outlets p o s i t i o n e d  100  at the end closest to the t u n n e l , a gas inlet at the far end o f the cage, a n d a gas s a m p l i n g tube inserted at the center to a depth o f 10 c m above the cage f l o o r . T h e air outlets were c o v e r e d w i t h m e s h to prevent the rats f r o m p u s h i n g their noses outside the test cage. T h e experimenter w a s c o n c e a l e d b e h i n d a b l i n d d u r i n g testing. T h e testing apparatus and O2 meter readout were v i d e o r e c o r d e d d u r i n g testing. D u r i n g b o t h t r a i n i n g and e x p e r i m e n t a l sessions, rats were first l o c k e d into the upper cage for 2 m i n . T h e l o c k w a s then r e m o v e d and they were able to enter the l o w e r test cage for a f o o d r e w a r d o f 2 0 H o n e y N u t C h e e r i o s ™ ( G e n e r a l M i l l s , Inc., M i n n e s o t a ) . W h e n the rat started eating the f o o d r e w a r d , gas f l o w (air, air w i t h p e p p e r m i n t or CO2) into the test cage w a s started at a rate o f 1 7 % o f the cage v o l u m e per m i n u t e . T h e session ended 3 0 0 s after l o c k r e m o v a l . T h e a n i m a l s were a l l o w e d to exit and re-enter the test cage throughout the test p e r i o d . A t the e n d o f the s e s s i o n , the r e m a i n i n g r e w a r d items were r e m o v e d and the rat w a s returned to the h o l d i n g room. F o r E x p e r i m e n t 1 the subjects were n i n e 5 - m o n t h o l d , m a l e W i s t a r rats. I n i t i a l l y they w e r e trained for 9 days to p e r f o r m the a p p r o a c h - a v o i d a n c e task w i t h air f l o w i n g into the test cage. T h i s t r a i n i n g w a s f o l l o w e d b y 17 days o f testing. T h e rats w e r e first tested over  five  c o n s e c u t i v e d a y s , w i t h air o n d a y 1, g r a d u a l - f i l l CO2 o n days 2 , 3 , and 4, a n d air a g a i n o n day 5. F r o m days 6 to 1 5 , the rats were tested t w o t i m e s w i t h air and w i t h static CO2 concentrations o f 5, 10, 15 and 2 0 % for a separate e x p e r i m e n t (Chapter 2). These data are not i n c l u d e d i n the current study, but served to p r o v i d e the rats w i t h further experience o f CO2 exposure. R a t s were then tested a g a i n w i t h g r a d u a l - f i l l CO2 and air o n days 16 and 17, r e s p e c t i v e l y . F o r E x p e r i m e n t 2 the subjects were seven 1 3 - m o n t h o l d , m a l e W i s t a r rats. B e f o r e this experiment,  they  had  performed  in  another  approach-avoidance  experiment,  and  had  c o n s i d e r a b l e experience w i t h air or CO2 f l o w i n g into the test cage. H o w e v e r they h a d not p r e v i o u s l y b e e n tested w i t h a n o v e l o d o u r s u c h as p e p p e r m i n t . T h e rats were tested o v e r 101  five  c o n s e c u t i v e d a y s , w i t h air o n day 1, air w i t h p e p p e r m i n t odour o n days 2 , 3 , and 4 , and air a g a i n o n day 5. F o r E x p e r i m e n t s 1 and 2 , w e recorded the total n u m b e r o f r e w a r d items eaten d u r i n g each test session, the latency to stop eating and the latency to leave the test cage after gas f l o w started. F o r E x p e r i m e n t 1 w e also recorded the 0  2  concentrations w h e n the rat stopped eating  a n d w h e n it left the test cage, and used these values to calculate CO2 concentrations as d e s c r i b e d above.  5.2.3 B e h a v i o u r a l Responses: E x p e r i m e n t 3 In this e x p e r i m e n t w e e x a m i n e d the b e h a v i o u r a l responses o f rats d u r i n g the gradual a d d i t i o n o f either CO2 or air w i t h p e p p e r m i n t odour. T h e exposure c h a m b e r w a s a 2 0 L p o l y p r o p y l e n e cage m e a s u r i n g 2 0 . 5 x 4 5 . 5 x 2 4 c m ( L a b P r o d u c t s Inc.), fitted w i t h a P l e x i g l a s l i d . T h e l i d h a d a gas inlet centered at the e n d , t w o air outlets p o s i t i o n e d at the opposite e n d , and a gas s a m p l i n g tube inserted at the center o f the c h a m b e r to a depth o f h a l f the c h a m b e r height. T h e air outlets were c o v e r e d w i t h m e s h to prevent the rats f r o m p u s h i n g their noses outside the c h a m b e r . T h e b a c k a n d sides o f the c h a m b e r were c o v e r e d w i t h b l a c k paper so that the a n i m a l s c o u l d not see the p e r s o n c o n d u c t i n g the e x p e r i m e n t . T h e subjects w e r e t h i r t y - t w o 4 to 6 - m o n t h - o l d , m a l e W i s t a r rats. A n i m a l s were r a n d o m l y a l l o c a t e d to the CO2 or air w i t h p e p p e r m i n t odour treatment groups (n = 16 for both). T h e rats were i n d i v i d u a l l y p l a c e d into the euthanasia c h a m b e r for a 2 7 - m i n p e r i o d o f a c c l i m a t i z a t i o n , d u r i n g w h i c h air w a s a d d e d to the c h a m b e r at a rate o f 1 7 % o f the c h a m b e r v o l u m e per m i n u t e . A f t e r a c c l i m a t i z a t i o n , air f l o w w a s stopped and either CO2 or air w i t h p e p p e r m i n t odour w a s started at a rate o f 17%> o f the c h a m b e r v o l u m e per m i n u t e . C O v t r e a t e d a n i m a l s r e m a i n e d i n the c h a m b e r a n d were m o n i t o r e d u n t i l death, but a n i m a l s treated w i t h p e p p e r m i n t odour w e r e 102  r e m o v e d f r o m the c h a m b e r at the e n d o f the 1 3 5 - s o b s e r v a t i o n p e r i o d . P r e l i m i n a r y observations s h o w e d that C02-treated a n i m a l s ceased a l l p u r p o s e f u l m o v e m e n t w i t h i n this p e r i o d , so a n y relevant effects o f p e p p e r m i n t exposure w o u l d be present d u r i n g this t i m e . The  euthanasia c h a m b e r a n d O2 meter readout  were  video  recorded  d u r i n g the  e x p e r i m e n t a l procedure. E a c h a n i m a l w a s s c o r e d c o n t i n u o u s l y d u r i n g the last 135 s o f the a c c l i m a t i z a t i o n p e r i o d (baseline) a n d f o r 135 s after gas f l o w b e g a n (exposure) f o r p r e - d e f i n e d behaviours thought to relate to distress (Table 1). In a p r e v i o u s study these behaviours were f o u n d to increase d u r i n g g r a d u a l - f i l l CO2 exposure (Chapter 2 ) .  5.2.4 S t a t i s t i c a l A n a l y s e s  5.2.4.1 Experiment 1 -Approach-avoidance Testing with CO2 Dependent variables were a n a l y z e d f o r the first three days o f CO2 exposure w i t h a m i x e d m o d e l ( S A S v 9 . 1 ) w h i c h i n c l u d e d rat as a r a n d o m effect (8 d.f), a n d tested f o r a linear effect o f order (1 d.f.) against a n error term w i t h 17 d.f. F o r those v a r i a b l e s where n o statistical differences w e r e f o u n d across the first three days o f CO2 exposure ( a l l except n u m b e r o f r e w a r d items eaten), data were averaged w i t h i n rat a n d the average response w a s then c o m p a r e d to the response o n the final day o f C 0 testing ( D a y 16) w i t h a m i x e d m o d e l w h i c h i n c l u d e d rat as a 2  r a n d o m effect (8 d.f.) a n d e x a m i n e d the effect o f order (1 d.f.) against an error term w i t h 8 d.f. N u m b e r o f r e w a r d items eaten w a s c o m p a r e d between the third a n d final CO2 test sessions w i t h a similar mixed model.  5.2.4.2 Experiment 2 - Approach-avoidance Testing with Peppermint Odour O n l y 1 o f the 7 rats ate f e w e r than 2 0 r e w a r d items d u r i n g testing w i t h a i r o r p e p p e r m i n t odour, so statistical analyses were not p e r f o r m e d w i t h this v a r i a b l e . T h e r e m a i n i n g dependent variables were c o m p a r e d across the three days o f testing w i t h p e p p e r m i n t o d o u r w i t h a m i x e d 103  m o d e l w h i c h i n c l u d e d rat as a r a n d o m effect (6 d.f) and tested for a linear effect o f order (1 d.f.) against a n error t e r m w i t h 13 d.f. R a t s s h o w e d s i m i l a r responses to p e p p e r m i n t o d o u r exposure o v e r the three days o f testing, so data were averaged w i t h i n rat for the t w o days o f testing w i t h air and for the three days o f testing w i t h p e p p e r m i n t odour. D e p e n d e n t variables were t h e n c o m p a r e d w i t h a m i x e d m o d e l w h i c h i n c l u d e d rat as a r a n d o m effect (6 d.f) and e x a m i n e d the effect o f gas treatment (1 d.f.) against an error t e r m w i t h 6 d.f.  5.2.4.3 Experiment 3 - Behavioural Responses T h e n u m b e r o f t i m e s the rat reared and the t i m e spent w i t h the nose i n contact w i t h the c h a m b e r l i d were a n a l y z e d w i t h a m i x e d m o d e l w h i c h i n c l u d e d rat as a r a n d o m effect (30 d.f.) and e x a m i n e d the effect o f p e r i o d (1 d . f ) , gas treatment ( l . d . f ) a n d the interaction b e t w e e n p e r i o d and gas (1 d.f.) against an error t e r m w i t h 3 0 d.f. E s c a p e b e h a v i o u r s and a c t i v i t y (recorded as side changes) were not o b s e r v e d i n a l l a n i m a l s , so the n u m b e r o f a n i m a l s w h i c h s h o w e d increases i n these b e h a v i o r s d u r i n g exposure w a s c o m p a r e d b e t w e e n gas treatments b y a G-test w i t h a W i l l i a m ' s c o r r e c t i o n (described i n S o k a l and R o l f , 1995).  5.3 Results 5.3.1 Experiment 1 - Approach-avoidance testing with CO2 D u r i n g a p p r o a c h - a v o i d a n c e testing o n the three c o n t r o l days w i t h air ( D a y s 1, 5 and 17), rats c o n s u m e d a l l 2 0 r e w a r d i t e m s , a n d , o n average ( m e a n ± S . E . ) , they stopped eating and left the test cage after 2 6 6 ± 4 s and 2 8 8 ± 2 s, respectively. In contrast, o n the four CO2 test days ( D a y s 2 , 3 , 4 and 16), rats c o n s u m e d an average o f o n l y 2.7 ± 0.2 r e w a r d i t e m s , and their latencies to stop eating a n d leave the test cage d r o p p e d to 30 ± 2 s and 4 0 ± 2 s, respectively.  104  A c r o s s the first three days o f testing w i t h CO2, there w a s n o change i n the latencies to stop eating o r leave the test cage, o r i n the CO2 c o n c e n t r a t i o n at w h i c h rats stopped eating a n d left the test cage ( F i g . 5.1 a , b; P > 0.1 f o r a l l ) . H o w e v e r , o n the final d a y o f testing rats s h o w e d a 5 2 % increase i n latency to stop eating  (Fi  >8  = 18.99, P < 0 . 0 0 5 ) a n d a 25%> increase i n latency  to leave the test cage (F\$ = 7 . 7 5 , P < 0.05), resulting i n higher CO2 concentrations at these t i m e p o i n t s (stop eating: F  ) ; 8  = 3 1 . 9 , P < 0 . 0 0 1 ; leave test cage: F % = 1 5 . 2 , P < 0.01). h  T h e n u m b e r o f r e w a r d items eaten s h o w e d a l i n e a r increase over the first three days o f testing w i t h CO2 ( F i g . 5.1 c ; F\ \i = 5 . 4 5 , P < 0.05). T h e n u m b e r o f r e w a r d items eaten also t  increased f r o m the t h i r d to the final test session (F\# = 2 5 . 5 4 , P < 0.001).  5.3.2 Experiment 2 - Approach-avoidance testing with peppermint odour R a t s ' p e r f o r m a n c e o n the a p p r o a c h - a v o i d a n c e task w a s s i m i l a r o n the t w o c o n t r o l days w i t h a i r ( D a y s 1 a n d 5 ) , a n d o n the three days o f testing w i t h p e p p e r m i n t o d o u r ( D a y s 2 , 3 a n d 4). D u r i n g testing w i t h b o t h a i r a n d p e p p e r m i n t odour, s i x o f the seven rats c o n s u m e d a l l 2 0 f o o d r e w a r d items o n e a c h test d a y . T h e seventh rat ate f o r the entire test p e r i o d e a c h d a y , but o n l y c o n s u m e d 17 to 2 0 o f . t h e r e w a r d items d u e to a s l o w eating rate. I n c o m p a r i s o n to a i r e x p o s u r e , p e p p e r m i n t exposure d i d not affect the latency f o r rats to stop eating ( 2 3 2 v s . 2 3 5 ± 14 s ; F  h6  = 0 . 6 6 , P > 0.1) or leave the test cage (274 v s . 2 8 3 ± 6 s; F\  fi  = 2.06, P > 0.1).  R a t s ' p e r f o r m a n c e w a s also consistent across the three days o f testing w i t h p e p p e r m i n t odour. T h e y c o n s u m e d a s i m i l a r n u m b e r o f r e w a r d i t e m s , a n d there w a s n o difference i n their latency to stop eating ( F  U  3  = 0 . 3 3 , P > 0.1) or to leave the test cage ( F  U  3  = 0.44, P > 0.1).  5.3.3 Experiment 3 - Behavioural responses D u r i n g the 1 3 5 - s baseline p e r i o d , rats f r o m b o t h the CO2 a n d the p e p p e r m i n t o d o u r treatment groups reared about f o u r t i m e s a n d spent about 12 to 14 s w i t h the nose t o u c h i n g the 105  c h a m b e r l i d ( F i g . 5.2). A f t e r i n i t i a t i o n o f either CO2 or p e p p e r m i n t o d o u r , rats s h o w e d increases i n b o t h rearing ( F i , 3 o  =  1 4 . 1 3 , P < 0 . 0 0 1 ) , and t i m e spent w i t h the nose t o u c h i n g the c h a m b e r l i d  (^i,30 = 8 . 1 1 , P < 0.01). W h i l e the increase i n response for these b e h a v i o u r s w a s n u m e r i c a l l y larger w i t h CO2 than w i t h p e p p e r m i n t o d o u r , w e f o u n d neither an effect o f gas treatment n o r an interaction b e t w e e n p e r i o d and gas treatment ( P > 0.1). D u r i n g exposure to either CO2 or p e p p e r m i n t o d o u r , less than h a l f o f the rats s h o w e d increases i n a c t i v i t y i n c o m p a r i s o n to baseline. F u r t h e r m o r e , the n u m b e r o f rats that s h o w e d a n increase d i d not d i f f e r between the CO2 (6 o f 16 rats) and p e p p e r m i n t o d o u r (7 o f 16 rats) treatment groups ( G = 0 . 1 2 , P > 0.1). D u r i n g the baseline p e r i o d , escape b e h a v i o u r s were o n l y p e r f o r m e d b y one rat. T h i s rat was  f r o m the p e p p e r m i n t o d o u r treatment g r o u p , and it d i d not p e r f o r m escape b e h a v i o u r s  d u r i n g exposure to p e p p e r m i n t odour. Increases i n escape b e h a v i o u r s w e r e o b s e r v e d i n 1 o f 16 rats d u r i n g p e p p e r m i n t exposure, but were o b s e r v e d i n 10 o f 16 rats d u r i n g CO2 exposure (G = 1 1 . 8 9 , P < 0 . 0 0 1 ) . T h e n u m b e r o f escape b e h a v i o u r s p e r f o r m e d b y rats d u r i n g CO2 exposure ranged f r o m 1 to 2 1 .  5.4 Discussion P r e v i o u s studies have f o u n d that w h e n rats are e x p o s e d to n o v e l s t i m u l i s u c h as n o v e l e n v i r o n m e n t s ( D u b o v i c k y et a l . , 1 9 9 9 ; M o n t g o m e r y , 1955) and objects ( Z a n g r o s s i and F i l e , 1994), they s h o w  signs o f h a b i t u a t i o n b y the s e c o n d exposure.  In  contrast, the rats  in  E x p e r i m e n t 1 d i d not s h o w any r e d u c t i o n i n their a v o i d a n c e o f g r a d u a l - f i l l CO2 exposure over the first three days o f a p p r o a c h - a v o i d a n c e testing. T h e y ate faster o n the s e c o n d and t h i r d days o f testing, but s h o w e d s i m i l a r latencies to stop eating a n d leave the test cage. B e c a u s e the response to CO2 d i f f e r e d f r o m that o b s e r v e d w i t h other s i m p l e , n o v e l s t i m u l i , it is u n l i k e l y that  106  the rats' response w a s due to novelty. T h i s c o n c l u s i o n is supported b y the results o f E x p e r i m e n t 2 , i n w h i c h n o v e l p e p p e r m i n t odour had n o effect o n rats' p e r f o r m a n c e d u r i n g a p p r o a c h a v o i d a n c e testing. H e n c e , exposure to a n o v e l o d o u r is not s u f f i c i e n t to deter rats f r o m this type o f task. F u r t h e r m o r e , b y the final d a y o f a p p r o a c h - a v o i d a n c e testing w i t h CO2, rats were s t i l l showing  consistent  aversion  to  CO2  concentrations  below  those  needed  to  cause  u n c o n s c i o u s n e s s . T h i s result indicates that rats do not habituate to g r a d u a l - f i l l CO2 exposure, and suggests that a v e r s i o n to CO2 is due m a i n l y to factors other t h a n n o v e l t y . R a t s i n the current study i n i t i a l l y left the test cage w h e n CO2 concentrations reached 1 4 % , but this increased to 18%) o n the final day o f testing. In p r e v i o u s a p p r o a c h - a v o i d a n c e studies o n CO2 a v e r s i o n i n rats, the rats were tested for a v e r s i o n to CO2 after b e i n g f a m i l i a r i z e d w i t h CO2 exposure (Chapters 3 and 4). T h e current results suggest that this l i k e l y resulted i n a modest u n d e r e s t i m a t i o n o f the CO2 concentrations that rats find a v e r s i v e o n i n i t i a l exposure. O n e potential e x p l a n a t i o n for this increased tolerance for CO2 w i t h repeated exposure is that the rats learned to tolerate the unpleasant sensations associated w i t h CO2 exposure, or that they learned strategies, s u c h as b r e a t h o l d i n g , that a l l o w e d t h e m to r e m a i n i n the test cage for longer. A l t e r n a t i v e l y , the rats m a y have d e v e l o p e d an increased p h y s i o l o g i c a l tolerance for CO2. P r e v i o u s studies have f o u n d that c h r o n i c exposure to elevated CO2 c a n result i n a reduced ventilatory  response to h y p e r c a p n i a t h r o u g h  a c i d - b a s e adjustments ( L a i  et a l . , 1981) or  alterations i n c h e m o r e c e p t o r a c t i v i t y ( M i t c h e l l a n d J o h n s o n , 2 0 0 3 ) . C h r o n i c exposure to l o w l e v e l s o f CO2 (<3%) has also been s h o w n to increase the l e v e l o f h y p e r c a p n i a needed to cause d y s p n e a d u r i n g acute CO2 exposure i n h u m a n s ( B l o c h - S a l i s b u r y et a l . , 1996). H o w e v e r , i n c o m p a r i s o n to other studies i n w h i c h this increased tolerance has b e e n demonstrated, rats i n the current study were e x p o s e d to CO2 for o n l y short periods ( less than 5 m i n per day), whereas changes i n d y s p n e a tolerance i n h u m a n s d e v e l o p e d o n l y after m u l t i p l e days o f c h r o n i c CO2 exposure ( B l o c h - S a l i s b u r y et a l . , 1996).  / 107  O n l y one study has l o o k e d at the effect o f short, d a i l y CO2 exposures o n ventilatory responses to a n acute CO2 c h a l l e n g e . W a t e r s a n d T i n w o r t h ( 2 0 0 1 ) e x a m i n e d changes i n the v e n t i l a t o r y responses o f piglets to a c h a l l e n g e w i t h a n i n s p i r e d gas m i x t u r e o f 9 % 0 CO2 after seven days o f acute, c y c l i c exposure to this same gas m i x t u r e . C y c l i c  2  and 6 % exposure  c o n s i s t e d o f one 4 8 - m i n session per day i n w h i c h fresh air and exposure to the gas m i x t u r e were alternated e v e r y 4 m i n . F o l l o w i n g seven days o f c y c l i c exposure, the piglets s h o w e d a s m a l l e r increase i n breathing rate and v o l u m e i n response to the c h a l l e n g e i n c o m p a r i s o n to controls. H o w e v e r , it is not k n o w n whether the r e l a t i v e l y short p e r i o d s o f CO2 exposure that o c c u r r e d d u r i n g the current study were sufficient to cause s u c h changes, and whether these changes w o u l d have altered the rats' a v e r s i o n to CO2 exposure. B e h a v i o u r a l changes o f rats d u r i n g f o r c e d exposure to an u n f a m i l i a r s t i m u l u s , s u c h as p e p p e r m i n t o d o u r , m i g h t be due to n o v e l t y , w h i c h w o u l d decrease w i t h repeated exposure, or due to i n t r i n s i c a v e r s i o n to the s t i m u l u s , w h i c h w o u l d r e m a i n constant w i t h repeated exposure. W h i l e h u m a n s do not generally f i n d p e p p e r m i n t o d o u r i n t r i n s i c a l l y unpleasant, n o p r e v i o u s studies have e x a m i n e d a v e r s i o n to p e p p e r m i n t o d o u r i n rats. In E x p e r i m e n t 2 w e f o u n d that rats s h o w e d n o s i g n o f a v e r s i o n to p e p p e r m i n t o d o u r at the concentrations u s e d i n the current study, e v e n o n the first exposure. T h i s result suggests that rats' a v e r s i o n to this s t i m u l u s w a s less than their m o t i v a t i o n to o b t a i n the r e w a r d items. N o t o n l y d i d rats c o n s u m e a s i m i l a r n u m b e r o f r e w a r d i t e m s i n a s i m i l a r a m o u n t o f t i m e w i t h b o t h air a n d p e p p e r m i n t o d o u r , but d u r i n g p e p p e r m i n t o d o u r exposure they r e m a i n e d i n the test cage for a n average o f 4 8 s after they stopped eating. T h e rats' w i l l i n g n e s s to r e m a i n i n the test cage e v e n after the r e w a r d w a s f i n i s h e d suggests that rats do not f i n d p e p p e r m i n t o d o u r a v e r s i v e , and that any b e h a v i o u r a l responses o b s e r v e d d u r i n g f o r c e d exposure were not due to a v e r s i o n . In a p r e v i o u s study w e f o u n d that exposure to a g r a d u a l l y i n c r e a s i n g c o n c e n t r a t i o n o f CO2 caused an increase i n b o t h e x p l o r a t o r y and escape b e h a v i o u r s i n rats (Chapter 2). O n e o f 108  the a i m s o f the current study w a s to determine whether these responses were due to n o v e l t y or other aversive properties o f CO2. In E x p e r i m e n t 3 , exposure to either CO2 or a n o v e l o d o u r i n g r a d u a l l y i n c r e a s i n g concentrations resulted i n increased a c t i v i t y , r e a r i n g , and t i m e spent w i t h the nose t o u c h i n g the c h a m b e r l i d . H o w e v e r , m o r e rats p e r f o r m e d escape b e h a v i o u r s d u r i n g CO2 exposure than d u r i n g exposure to p e p p e r m i n t odour. T h i s indicates that  exploratory  b e h a v i o u r s d u r i n g g r a d u a l - f i l l CO2 exposure c o u l d be due to n o v e l t y , but that escape b e h a v i o u r s are m a i n l y due to other properties o f CO2. E x p l o r a t o r y b e h a v i o u r s are d i f f i c u l t to interpret i n terms o f a n i m a l distress, but escape b e h a v i o u r s p r e s u m a b l y indicate that the a n i m a l w o u l d exit the exposure c h a m b e r i f g i v e n the opportunity. These results therefore suggest that distress d u r i n g g r a d u a l - f i l l CO2 exposure is not due to n o v e l t y . H o w e v e r , one a n i m a l d i d p e r f o r m escape b e h a v i o u r s i n response to p e p p e r m i n t odour exposure, so exposure to a n o v e l s t i m u l u s m a y also contribute to distress d u r i n g CO2 exposure. P r e v i o u s studies h a v e f o u n d that f r e e z i n g  in  response to a n o v e l o d o u r depends o n the o d o u r that is u s e d and its intensity ( W a l l a c e and R o s e n , 2 0 0 0 ) , so w e cannot rule out the p o s s i b i l i t y that another o d o u r s t i m u l u s m i g h t e l i c i t a s i m i l a r b e h a v i o u r a l response to that seen d u r i n g CO2 exposure. O f the three potential causes o f distress and a v e r s i o n d u r i n g CO2 exposure - n o v e l t y , p a i n and d y s p n e a - the current study indicates that n o v e l t y l i k e l y contributes to the responses o f rats d u r i n g g r a d u a l - f i l l CO2 exposure, but it is not the m a i n cause o f distress and a v e r s i o n . M o r e o v e r , p r e v i o u s studies have f o u n d that b e h a v i o r a l responses and a v e r s i o n to a g r a d u a l l y i n c r e a s i n g c o n c e n t r a t i o n o f CO2 o c c u r at concentrations that are b e l o w the threshold for p a i n at the eyes a n d the nasal m u c o s a (Chapters 2 , 3 and 4). T h e r e f o r e , the m o s t l i k e l y cause o f distress and a v e r s i o n d u r i n g g r a d u a l - f i l l CO2 exposure is d y s p n e a , w h i c h o c c u r s i n h u m a n s at CO2 concentrations o f o n l y 8 % ( D r i p p s & C o m r o e , 1 9 4 7 ; L i o t t i et a l . , 2 0 0 1 ) . Further research is needed to determine whether d y s p n e a does o c c u r i n rats i n response to CO2 exposure, and i f so whether it is p o s s i b l e to mitigate sensations o f d y s p n e a d u r i n g CO2 euthanasia. 109  T a b l e 5;1. D e s c r i p t i o n s o f rat behaviours r e c o r d e d d u r i n g baseline and d u r i n g exposure to CO2 or p e p p e r m i n t o d o u r ( E x p e r i m e n t 3). Behaviour  Description  Activity  M o v e m e n t that results i n the b a c k feet c r o s s i n g a l i n e that d i v i d e s the length o f the c h a m b e r i n h a l f (event).  Rear  R a i s i n g the upper b o d y  w h i l e standing o n the b a c k feet.  Includes w a l l c l i m b i n g . C l i m b i n g o n the air s a m p l i n g tube w h i l e c h e w i n g it and r e a r i n g d u r i n g g r o o m i n g were e x c l u d e d (event). N o s e to l i d  T i m e spent w i t h the nose i n contact w i t h the c h a m b e r l i d (state).  Escape behaviours: S c r a t c h at l i d  A r a p i d m o v e m e n t o f the front p a w f r o m the l i d t h r o u g h at least a 90° d o w n w a r d angle (event).  P u s h at l i d  A  p u s h at the c h a m b e r l i d u s i n g the nose or front  e v i d e n c e d b y b o d y and l i d m o v e m e n t (event).  110  paw  a) 50 40 30 20 • Leave • Stop eating  10 0  final  20  n  15  M 10 -2  5  • Leave • Stop eating  final  2 3 Exposure number  final  Figure 5.1. A p p r o a c h - a v o i d a n c e responses to CO2 for the first three days and for the f i n a l day ( D a y 16) o f exposure ( E x p e r i m e n t 1). M e a n ( ± S E M ) (a) latency to stop eating and leave the test cage, (b) CO2 c o n c e n t r a t i o n w h e n rats stopped eating and left the test cage, a n d (c) n u m b e r o f r e w a r d items eaten (n = 9 rats).  Ill  F i g u r e 5.2.  B e h a v i o u r a l responses o f rats to CO2 euthanasia and p e p p e r m i n t o d o u r exposure  ( E x p e r i m e n t 3). L e a s t squares m e a n ( ± S E M ) (a) n u m b e r o f rears, and (b) t i m e spent w i t h the nose i n contact w i t h the test cage l i d d u r i n g baseline and d u r i n g exposure to either CO2 (n = 16 rats) or air w i t h p e p p e r m i n t odour (n = 16 rats).  112  5.5 References A n t o n , F., E u c h n e r , I., H a n d w e r k e r , H . O . , 1992. P s y c h o p h y s i c a l e x a m i n a t i o n o f p a i n i n d u c e d b y d e f i n e d CO2 pulses a p p l i e d to the nasal m u c o s a . P a i n 4 9 , 5 3 - 6 0 . A n t o n , F., P e p p e l , P., E u c h n e r ,  I.,  Handwerker,  H.O.,  1991. Controlled noxious  chemical  s t i m u l a t i o n : responses o f rat t r i g e m i n a l b r a i n s t e m neurones to CO2 pulses a p p l i e d to the nasal m u c o s a . N e u r o s c i . Lett. 1 2 3 , 2 0 8 - 2 1 1 . B l o c h - S a l i s b u r y , E., S h e a , S . A . , B r o w n , R., E v a n s , K . , B a n z e t t , R . B . , 1996. A i r hunger i n d u c e d b y acute increase i n Pco2 adapts to c h r o n i c e l e v a t i o n o f Pco2 i n ventilated h u m a n s . J . A p p l . Physiol. 81,949-956. Cain, W.S., Murphy, C L .  1 9 8 0 . Interaction between c h e m o r e c e p t i v e m o d a l i t i e s o f o d o u r a n d  irritation. N a t u r e . 2 8 4 , 2 5 5 - 2 5 7 . C h e n , X . , G a l l a r , J . , P o z o , M . A . , B a e z a , M . , B e l m o n t e , C , 1 9 9 5 . CO2 s t i m u l a t i o n o f the c o r n e a : a c o m p a r i s o n b e t w e e n h u m a n sensation and nerve a c t i v i t y  in polymodal  nociceptive  afferents o f the cat. E u r . J . N e u r o s c i . 7, 1 1 5 4 - 1 1 6 3 . C o e n e n , A . M . , D r i n k e n b u r g , W . H . , H o e n d e r k e n , R., v a n L u i j t e l a a r , G . L . , 1 9 9 5 . C a r b o n d i o x i d e euthanasia i n rats: o x y g e n s u p p l e m e n t a t i o n m i n i m i z e s signs o f agitation and a s p h y x i a . L a b . A n i m . 29, 262-268. D r i p p s , R . D . , C o m r o e , J . H . , 1 9 4 7 . T h e respiratory and c i r c u l a t o r y response o f n o r m a l m a n to i n h a l a t i o n o f 7.6 a n d 10.4 per cent CO2 w i t h a c o m p a r i s o n o f the m a x i m a l v e n t i l a t i o n produced  by  severe  muscular  exercise,  inhalation  of  CO2  and  maximal  voluntary  h y p e r v e n t i l a t i o n . A m . J . P h y s i o l . 149, 4 3 - 5 1 . Dubovicky,  M.,  Skultetyova,  I.,  Jezova,  D.,  1 9 9 9 . N e o t n a t a l stress alters habituation  of  e x p l o r a t o r y b e h a v i o u r i n adult m a l e but not f e m a l e rats. P h a r m a c o l . B i o c h e m . B e . 6 4 , 6 8 1 686.  113  F e n g , Y . , S i m p s o m , T. L., 2 0 0 3 . N o c i c e p t i v e sensation and sensitivity e v o k e d  from  human  c o r n e a and c o n j u n c t i v a s t i m u l a t e d b y CO2. Invest. O p h t h . V i s . S c i . 4 4 , 5 2 9 - 5 3 2 . L a i , Y . L . , L a m m , W . J . E , H i l d e b r a n d t , J . , 1 9 8 1 . V e n t i l a t i o n d u r i n g p r o l o n g e d h y p e r c a p n i a i n the rat. J . A p p l . P h y s i o l . 5 1 , 7 8 - 8 3 . L e a c h , M . C . , B o w e l l , V . A . , A l l a n , T . F . , M o r t o n , D . B . , 2 0 0 2 . A v e r s i o n to gaseous euthanasia agents i n rats and m i c e . C o m p a r a t i v e M e d . 5 2 , 2 4 9 - 2 5 7 . Liotti, M . , Brannan, S., Egan, G.,  Shade, R.,  M a d d e n , L.,  Abplanalp, B., Robillard,  R.,  L a n c a s t e r , J . , Z a m a r r i p a , F . E . , F o x , P.T., D e n t o n , D . , 2 0 0 1 . B r a i n responses associated w i t h c o n s c i o u s n e s s o f breathlessness (air hunger). P r o c . N a t . A c a d . S c i . 9 8 , 2 0 3 5 - 2 0 4 0 . M i t c h e l l , G . S . , J o h n s o n , S . M . , 2 0 0 3 . N e u r o p l a s t i c i t y i n respiratory m o t o r c o n t r o l . J . A p p l . Physiol. 94, 358-374. Montgomery,  K.C.  1955. The  relation b e t w e e n  fear  induced  by  novel  stimulation  and  exploratory b e h a v i o r . J . C o m p . P h y s i o l . P s y c h o l . 4 8 , 2 5 4 - 2 6 0 . P e p p e l , P., A n t o n , F., 1 9 9 3 . R e s p o n s e s o f rat m e d u l l a r y dorsal h o r n neurons f o l l o w i n g intranasal n o x i o u s c h e m i c a l s t i m u l a t i o n : effects o f s t i m u l u s intensity, d u r a t i o n , a n d i n t e r s t i m u l u s interval. J. Neurophysiol. 70, 2260-2275. S o k a l , R . R . , R o h l f , F.J., 1995. B i o m e t r y : the p r i n c i p l e s and practice o f statistics i n b i o l o g i c a l research, F r e e m a n , N e w Y o r k , p p . 7 2 8 - 7 3 2 . S m i t h , W . , H a r r a p , S . B . , 1 9 9 7 . B e h a v i o u r a l and c a r d i o v a s c u l a r responses o f rats to euthanasia u s i n g c a r b o n d i o x i d e gas. L a b . A n i m . 3 1 , 3 3 7 - 3 4 6 . T h u r a u f , N . , G u n t h e r , M . , P a u l i , E., K o b a l , G . , 2 0 0 2 . S e n s i t i v i t y o f the negative m u c o s a l potential to the t r i g e m i n a l target s t i m u l u s CO2. B r a i n R e s . 9 4 2 , 2 7 - 8 6 . W a l l a c e , K . J . , R o s e n , J . B . 2 0 0 0 . Predator odor as a n u n c o n d i t i o n e d fear s t i m u l u s i n rats: e l i c i t a t i o n o f f r e e z i n g b y t r i m e t h y l t h i a z o l i n e , a c o m p o n e n t o f f o x feces. B e h a v . N e u r o s c i . 114,912-922. 114  W a t e r s , K . A . , T i n w o r t h , K . D . , 2 0 0 1 . D e p r e s s i o n o f ventilatory responses after d a i l y c y c l i c h y p e r c a p n i c h y p o x i a i n piglets. J . A p p l . P h y s i o l . 9 0 , 1 0 6 5 - 1 0 7 3 . Youngentob,  S.L., Hornung,  D.E.,  M o z e l l , M . M . , 1991. Determination of carbon  dioxide  detection thresholds i n trained rats. P h y s i o l . . B e h a v . 4 9 , 2 1 - 2 6 . Zangrossi, H.,  File, S.E.,  1 9 9 2 . B e h a v i o r a l consequences i n a n i m a l tests o f anxiety  e x p l o r a t i o n o f exposure to cat odor. B r a i n R e s . B u l l . 2 9 , 3 8 1 - 3 8 8 .  115  and  CHAPTER 6: General Discussion T h e t e r m euthanasia refers to a g o o d death, w h i c h i m p l i e s a l a c k o f distress. H o w e v e r , due to the potential for CO2 to cause p a i n and d y s p n e a , there has been c o n s i d e r a b l e debate as to whether CO2 euthanasia c a n p r o d u c e death w i t h o u t distress. T h e t w o m a i n objectives o f m y thesis w e r e : 1) to determine whether g r a d u a l - f i l l CO2 euthanasia causes distress i n laboratory rats b y e x a m i n i n g b e h a v i o u r a l responses d u r i n g euthanasia, and a v e r s i o n d u r i n g a p p r o a c h a v o i d a n c e testing, a n d 2) to determine whether p a i n , d y s p n e a and n o v e l t y are l i k e l y sources o f distress d u r i n g this procedure.  6.1 Distress in rats during C 0 euthanasia 2  A s d i s c u s s e d i n C h a p t e r 1, the c o n c l u s i o n s o f p r e v i o u s research o n distress i n rats d u r i n g CO2 euthanasia have been h i g h l y v a r i a b l e . S o m e studies have f o u n d b e h a v i o u r a l responses to CO2 exposure that m a y be i n d i c a t i v e o f distress ( B r i t t , 1 9 8 6 ; C o e n e n et a l . , 1 9 9 5 ; I w a r s s o n and R e h b i n d e r , 1993), but others have not noted these effects ( B l a c k s h a w et a l . , 1 9 8 8 ; H a c k b a r t h et a l . , 2 0 0 0 ; H o r n e t t and H a y n e s , 1 9 8 4 ; S m i t h and H a r r a p , 1997). In a m o r e recent study, L e a c h et a l . (2002) f o u n d that CO2 causes a v o i d a n c e i n rats, suggesting that CO2 exposure is aversive. T h e results o f m y thesis b u i l d o n this p r e v i o u s research, and p r o v i d e further evidence that g r a d u a l - f i l l CO2 exposure does cause distress i n laboratory rats. Chapters 2 a n d 5 demonstrate that t w o w i d e l y u s e d strains o f laboratory rats, W i s t a r s a n d S p r a g u e - D a w l e y s , e x h i b i t behaviours that are i n d i c a t i v e o f distress d u r i n g g r a d u a l - f i l l CO2 euthanasia. D u r i n g CO2 exposure, b o t h strains s h o w e d e v i d e n c e o f increased e x p l o r a t i o n , i n c l u d i n g increased a c t i v i t y , rearing, and t i m e spent w i t h the nose t o u c h i n g the c h a m b e r l i d . M o r e i m p o r t a n t l y , rats i n b o t h studies s h o w e d scratching and p u s h i n g at the c h a m b e r l i d , b e h a v i o u r s that suggest the rats were t r y i n g to escape f r o m the ch am ber. Increased 116  exploratory  b e h a v i o u r has also b e e n observed i n s o m e p r e v i o u s studies (e.g. B r i t t , 1986), and w h i l e these b e h a v i o u r s indicate increased arousal they do not necessarily suggest distress. H o w e v e r , escape behaviours  strongly  suggest  that the  animals w o u l d  avoid  CO2  exposure  if given  the  o p p o r t u n i t y , and that f o r c e d CO2 exposure causes some l e v e l o f distress. C h a p t e r s 3 , 4 , and 5 demonstrate that rats w i l l a v o i d CO2 concentrations necessary to cause u n c o n s c i o u s n e s s , e v e n w h e n this requires that they g i v e u p a v a l u a b l e f o o d r e w a r d . In C h a p t e r 3 , I f o u n d that rats tolerated extended exposure to static CO2 concentrations o f 5 a n d 1 0 % , but that the latency to leave the test cage d r o p p e d d r a m a t i c a l l y at 1 5 % CO2. R a t s left the test cage at about this same concentration w h e n e x p o s e d to a g r a d u a l - f i l l procedure. In C h a p t e r 4 , I f o u n d that the f l o w rate used d u r i n g the g r a d u a l - f i l l procedure h a d o n l y a s m a l l effect o n a v o i d a n c e ; rats left the test cage at CO2 concentrations b e t w e e n 13 a n d 1 6 % w i t h f l o w rates r a n g i n g f r o m 3 to 2 7 % o f the test cage v o l u m e per m i n u t e . In C h a p t e r 5 , I f o u n d that rats are averse to CO2 regardless o f h a b i t u a t i o n , a l t h o u g h tolerance does increase s l i g h t l y w i t h repeated exposure.  This  increased tolerance  suggests that the aversive  concentrations  reported  in  C h a p t e r s 3 and 4 w o u l d l i k e l y have been about 4 % l o w e r o n i n i t i a l contact. Together, these results indicate that rats are averse to CO2 concentrations greater t h a n a p p r o x i m a t e l y 15%> regardless o f h o w CO2 is d e l i v e r e d . F r o m these results, I c o n c l u d e that f o r c e d exposure to h i g h e r concentrations l i k e l y results i n distress.  6.2 Sources of distress In C h a p t e r 1 , 1 i d e n t i f i e d three potential sources o f distress d u r i n g CO2 euthanasia: p a i n , d y s p n e a and n o v e l t y . T h e occurrence o f p a i n and d y s p n e a were not assessed d i r e c t l y i n the current thesis. H o w e v e r , I determined the CO2 concentrations that e l i c i t e d b e h a v i o u r a l responses a n d a v e r s i o n i n C h a p t e r s 2 , 3 , and 4, and c o m p a r e d these data w i t h p r e v i o u s research o n  117  n o c i c e p t o r a c t i v a t i o n i n rats, a n d p a i n and d y s p n e a i n h u m a n s . T h e potential for CO2 to e l i c i t distress as a result o f n o v e l t y w a s e x a m i n e d d i r e c t l y i n C h a p t e r 5.  6.2.1 Pain A s d i s c u s s e d i n C h a p t e r 1, CO2 i s k n o w n to cause p a i n at the n a s a l m u c o s a and c o r n e a i n h u m a n s at concentrations o f 3 0 to 5 0 % , and this c o n c e n t r a t i o n range is s i m i l a r to that required to stimulate n o c i c e p t o r s i n rat nasal m u c o s a ( A n t o n et a l . , 1 9 9 2 ; C h e n et a l . , 1 9 9 5 ; D a n n e m a n et a l . , 1 9 9 7 ; F e n g and S i m p s o n , 2 0 0 3 ; P e p p e l and A n t o n , 1 9 9 3 ; T h u r a u f et a l . , 2 0 0 2 ) . In C h a p t e r 2 , I f o u n d that rats started to s h o w b e h a v i o u r a l responses to g r a d u a l - f i l l CO2 euthanasia at CO2 concentrations o f o n l y 5 % CO2, and this response h a d p e a k e d and w a s d e c l i n i n g at CO2 concentrations o f o n l y 28%). S i m i l a r l y , i n Chapters 3 a n d 4 I f o u n d that rats a v o i d e d CO2 concentrations greater than a p p r o x i m a t e l y 1 5 % , and the m a x i m u m CO2 concentration tolerated b y a s i n g l e rat d u r i n g g r a d u a l - f i l l exposure w a s o n l y 2 5 % . T h e s e results suggest that p a i n does not account for the b e h a v i o u r a l responses o f rats d u r i n g g r a d u a l - f i l l CO2 euthanasia or for their a v e r s i o n to CO2. F u r t h e r m o r e , i n Chapter 2 I d i d not observe an increase i n p a i n - r e l a t e d b e h a v i o u r s , s u c h as h e a d - s h a k i n g and f a c e - w a s h i n g , d u r i n g CO2 e x p o s u r e , suggesting that the rats d i d not experience p a i n w h i l e they were able to m o u n t a b e h a v i o u r a l response. H o w e v e r , it is p o s s i b l e that rats experience p a i n around the t i m e o f loss o f posture.  6.2.2 Dyspnea A s d i s c u s s e d i n C h a p t e r 1, spontaneously breathing h u m a n s report d y s p n e a w i t h CO2 concentrations o f o n l y 8 % ( D r i p p s and C o m r o e , 1 9 4 7 ; L i o t t i et a l . , 2 0 0 1 ) , and severe d y s p n e a has b e e n reported w i t h CO2 concentrations o f 1 5 % to 20%) CO2 ( r e v i e w e d b y H i l l and F l a c k , 1908). W h i l e c o n c l u s i v e e v i d e n c e o f d y s p n e a i n rats is not a v a i l a b l e , the CO2 concentrations that e l i c i t d y s p n e a i n h u m a n s are consistent w i t h the concentrations that e l i c i t e d a b e h a v i o u r a l 118  responses and a v e r s i o n i n , laboratory rats, suggesting that d y s p n e a is a l i k e l y cause o f distress d u r i n g CO2 exposure. H u m a n s generally s h o w a delay b e t w e e n a change i n i n s p i r e d CO2 and the onset o f d y s p n e a , but v e r y little delay w a s observed i n rats. D u r i n g a p p r o a c h - a v o i d a n c e testing w i t h 2 0 % CO2, one rat refused to enter the test cage, and the latency to leave the test cage for the r e m a i n i n g rats ranged f r o m 2 to 16 s. If w e assume a m a x i m u m o f 2 s o f exposure i n the tunnel l e a d i n g to the test c a g e , this indicates that a v o i d a n c e o c c u r r e d after o n l y 2 s o f exposure for the rat that d i d not enter the test cage, and 18 s o f exposure for the rat w i t h the longest latency to leave the test cage. Part o f the d e l a y before onset o f d y s p n e a is due to the t i m e taken for i n s p i r e d C 0  2  to  increase b l o o d CO2 l e v e l s , and stimulate peripheral and central c h e m o r e c e p t o r s i n the c a r o t i d b o d i e s and the m e d u l l a , respectively. F o r h u m a n s , the delay b e t w e e n a change i n i n s p i r e d C 0  2  and the onset o f increased v e n t i l a t i o n is 5 to 1 5 s d e p e n d i n g o n w h e t h e r peripheral or central chemoreceptors are d r i v i n g the response ( r e v i e w e d b y  C u n n i n g h a m et a l . , 1986).  While  responses to h y p e r c a p n i a tend to o c c u r m a i n l y v i a the central c h e m o r e c e p t o r s , for w h i c h there is a l o n g e r d e l a y , the p e r i p h e r a l chemoreceptors also contribute. B l o o d CO2  concentrations  c o n t i n u e to increase towards i n s p i r e d l e v e l s , r e s u l t i n g i n increased v e n t i l a t i o n and d y s p n e a i f h y p e r c a p n i a is sufficient. B a n z e t t (1996) c a l c u l a t e d that a step change i n i n s p i r e d CO2 against a h y p o x i c b a c k g r o u n d results i n a l o g a r i t h m i c increase i n d y s p n e i c sensations i n h u m a n s , w i t h a h a l f - t i m e o f a p p r o x i m a t e l y 32 s for d e v e l o p m e n t o f a stable d y s p n e a rating. T h e t i m e to onset o f d y s p n e a w a s not reported, but the h a l f - t i m e p r o v i d e s an i n d i c a t i o n o f h o w l o n g it t o o k f o r moderate l e v e l s o f d y s p n e a to d e v e l o p under the c o n d i t i o n s that were used. S i m i l a r l y , H a l d a n e a n d S m i t h ( r e v i e w e d b y H i l l and F l a c k , 1908) f o u n d that there w a s a d e l a y o f 1 to 2 m i n u n t i l severe d y s p n e a set i n d u r i n g i n h a l a t i o n o f 18.6%> CO2. H o w e v e r , rats m a y d i f f e r f r o m h u m a n s i n the t i m e taken to d e v e l o p dyspnea. L a g n e a u x (1986) f o u n d that rats s h o w o n l y a 2 s l a g i n their 119  ventilatory response to inspiration of 1.5% CO2, suggesting a much shorter circulatory delay in rats. Furthermore, the increase in ventilation occurred much more quickly with 1.5% CO2 than with 0.5 or 1% CO2, indicating that the time for development of ventilation, and likely dyspnea, is dependent on CO2 concentration. These results suggest that the distress and aversion observed in this thesis could have been due to dyspnea.  6.2.3 Novelty The results o f Chapter 5 illustrate the role of novelty on rat responses to gas exposure. While novelty results in increased exploratory behaviours and may contribute to distress due to fear, it does not account for performance of escape behaviours or avoidance o f CO2. These results suggest that novelty is not a major source o f distress during gradual-fill CO2 euthanasia.  6.2.4 Alternative hypotheses One alternative explanation for behavioural signs of distress and aversion in rats during CO2 exposure is that they could detect the onset bf unconsciousness, and that this sensation o f diminished consciousness  either evoked an innate escape response  or was perceived as  unpleasant. However, pigs, poultry and humans have been shown to lose consciousness during exposure to low O2 concentrations without demonstrating obvious attempts at avoidance (Cable, 2003; Raj & Gregory, 1995; Raj, 1996; Webster & Fletcher, 2004), suggesting that diminished consciousness on its own does not evoke escape responses or cause unpleasant sensations in other species. Furthermore, recent results from our research group suggest that avoidance of CO2 during gradual-fill exposure is not well correlated with ataxia, an initial indicator of diminished consciousness (Kirkden, unpublished data). Leach et al. (2002a) also found that rats avoided 25.5%) CO2 after only 1.1s, even though ataxia took 30 s to occur at this concentration  ofCOi. 120  A v o i d a n c e o f CO2 d u r i n g a p p r o a c h - a v o i d a n c e testing c o u l d also have b e e n i n f l u e n c e d b y the effects o f CO2 o n taste. T h e a p p r o a c h - a v o i d a n c e task that I used i s dependent o n rats b e i n g h i g h l y m o t i v a t e d to o b t a i n a f o o d r e w a r d , a n d alterations i n taste c o u l d affect this m o t i v a t i o n . CO2 f o r m s c a r b o n i c a c i d w h e n c o m b i n e d w i t h water, as w o u l d o c c u r at the o r a l m u c o s a , a n d acids are k n o w n to stimulate taste buds to p r o d u c e a sour taste ( D e S i m o n e et a l . , 2 0 0 1 ) . CO2 i s w i d e l y d e s c r i b e d to have a sour taste, but n o i n f o r m a t i o n i s a v a i l a b l e o n the c o n c e n t r a t i o n o f CO2 that w o u l d be necessary to e v o k e this taste i n rats. H o w e v e r , rats appeared h i g h l y m o t i v a t e d to eat right u n t i l they left the test cage, a n d w o u l d often grab one or t w o r e w a r d i t e m s to take w i t h t h e m . F u r t h e r m o r e , this hypothesis cannot account f o r the escape b e h a v i o u r s that rats p e r f o r m e d d u r i n g g r a d u a l - f i l l CO2 euthanasia.  6.2.5 Conclusions on sources of distress and aversion T h e m o s t l i k e l y source o f distress a n d a v e r s i o n d u r i n g g r a d u a l - f i l l CO2 exposure appears to be d y s p n e a . W h i l e there i s some q u e s t i o n as to whether this sensation c o u l d d e v e l o p w i t h i n the t i m e it t o o k f o r rats to e x h i b i t a v e r s i o n to CO2, the other hypotheses presented d o not f u l l y account f o r the responses that were observed. In order to determine m o r e c o n c l u s i v e l y whether CO2  causes  dyspnea  i n rats,  further  studies  are needed.  In h u m a n s , d y s p n e a  due to  e x p e r i m e n t a l l y - i n d u c e d h y p e r c a p n i a a n d due to disease has b e e n s h o w n to be r e l i e v e d b y i n h a l a t i o n o f a e r o s o l i z e d f u r o s e m i d e ( M i n o w a et a l . , 2 0 0 2 ; N i s h i n o et a l . , 2 0 0 0 ; O n g et a l . , 2 0 0 4 ; S h i m o y a m a a n d S h i m o y a m a , 2 0 0 2 ) . F u r o s e m i d e i s l a b e l l e d f o r use as a d i u r e t i c , a n d the m e c h a n i s m f o r its effects o n d y s p n e a i s not f u l l y understood. It has b e e n f o u n d to increase the a c t i v i t y o f p u l m o n a r y stretch receptors ( S u d o et a l . , 2 0 0 0 ) , w h i c h m a y f e e d b a c k into systems r e s p o n s i b l e f o r the generation o f d y s p n e a . T h i s effect o n p u l m o n a r y stretch receptors i s s p e c i f i c to aerosol d e l i v e r y a n d does not o c c u r after s y s t e m i c d e l i v e r y , suggesting that this is a l o c a l i z e d effect at the l e v e l o f the respiratory e p i t h e l i u m . A n e x p e r i m e n t to test the effects o f f u r o s e m i d e 121  o n tolerance to CO2 w o u l d p r o v i d e m o r e direct evidence o f the role o f d y s p n e a i n rat a v e r s i o n to C0 . 2  6.3 Critique of methods To  e x a m i n e distress associated w i t h CO2 euthanasia i n rats I u s e d t w o  different  m e t h o d s : 1) b e h a v i o u r a l assessment o f rat responses d u r i n g CO2 euthanasia, and 2) a p p r o a c h a v o i d a n c e testing w i t h static and g r a d u a l l y i n c r e a s i n g concentrations o f CO2. W h i l e p r e v i o u s studies have assessed the b e h a v i o u r o f rats d u r i n g CO2 euthanasia a n d f o u n d v a r i a b l e results, I i m p r o v e d u p o n these studies b y d e v e l o p i n g a w e l l d e f i n e d list o f p a i n and distress b e h a v i o u r s , u s i n g an adequate s a m p l e size for statistics, a c c l i m a t i z i n g the rats before gas exposure, and c o m p a r i n g b e h a v i o u r a l responses to b o t h baseline levels and responses d u r i n g air exposure. Preference testing h a d also been u s e d to e x a m i n e rats' a v o i d a n c e o f CO2 ( L e a c h et a l . , 2 0 0 2 a ) , but I i m p r o v e d u p o n this m e t h o d o l o g y b y e x a m i n i n g the strength o f rats' m o t i v a t i o n to a v o i d CO2 u s i n g a p p r o a c h - a v o i d a n c e testing. W h i l e this m e t h o d h a d been u s e d to e x a m i n e gas a v e r s i o n i n other species (e.g. C o o p e r et a l . , 1 9 9 8 ; G e r r i t z e n et a l . , 2 0 0 0 ; R a j , 1 9 9 6 ; R a j  &  G r e g o r y , 1 9 9 5 ; W e b s t e r & F l e t c h e r , 2 0 0 4 ) , it h a d not p r e v i o u s l y been d e v e l o p e d for use w i t h rats. B o t h o f the assessment methods that I used p r o v i d e d  useful and  complementary  i n f o r m a t i o n about the potential for CO2 to e l i c i t distress. R a t s p e r f o r m e d escape behaviours d u r i n g CO2 euthanasia, and left the test cage before loss o f c o n s c i o u s n e s s d u r i n g a p p r o a c h avoidance  testing.  Both  o f these responses  suggest  that  gradual-fill  CO2  exposure  was  unpleasant a n d that f o r c e d exposure l i k e l y causes distress. H o w e v e r , rats' b e h a v i o u r a l responses d u r i n g CO2 euthanasia were not c o m p a r e d to a k n o w n aversive s t i m u l u s , so it is d i f f i c u l t to determine the l e v e l o f distress that is i n d i c a t e d b y the l e v e l o f responses that I o b s e r v e d . In fact,  122  one rat s h o w e d escape b e h a v i o u r s d u r i n g exposure to p e p p e r m i n t o d o u r , a s t i m u l u s that w a s f o u n d to be n o n - a v e r s i v e d u r i n g a p p r o a c h - a v o i d a n c e testing. T h i s f i n d i n g suggests that escape b e h a v i o u r s m a y not a l w a y s indicate a h i g h l e v e l o f distress. A p p r o a c h - a v o i d a n c e provides  a better i n d i c a t i o n o f the severity  o f distress caused b y CO2 exposure,  testing because  m o t i v a t i o n to a v o i d CO2 is c o m p a r e d against m o t i v a t i o n to o b t a i n a f o o d r e w a r d . A s d i s c u s s e d i n C h a p t e r 3 , rats' m o t i v a t i o n to obtain sweet f o o d s w h e n f e d ad l i b i t u m is moderate to h i g h ; therefore, a p p r o a c h - a v o i d a n c e testing indicates that their m o t i v a t i o n to a v o i d CO2 is at least moderate. A n e v e n better i n d i c a t i o n o f strength o f a v e r s i o n to CO2 c a n be g a i n e d b y f o o d d e p r i v i n g rats f o r different periods o f t i m e p r i o r to testing to ensure that their m o t i v a t i o n for the f o o d r e w a r d is h i g h . U s i n g this procedure, K i r k d e n et a l . (2005) f o u n d that even after f o o d d e p r i v a t i o n for up to 24 h, rats s h o w e d m u c h the same a v e r s i o n to g r a d u a l - f i l l CO2 exposure. T h i s result suggests that rats find this exposure h i g h l y aversive. A p p r o a c h - a v o i d a n c e testing appears to be a m o r e sensitive measure o f a v e r s i o n to CO2 i n rats than s i m p l e b e h a v i o u r a l responses. A l t h o u g h rats s h o w e d m a n y b e h a v i o u r a l changes d u r i n g CO2 euthanasia, the m a j o r i t y o f the b e h a v i o u r s observed are d i f f i c u l t to interpret. O n l y escape b e h a v i o u r s  provide  a clear i n d i c a t i o n o f a v e r s i o n . B e h a v i o u r a l responses to  CO2  euthanasia were also h i g h l y v a r i a b l e b e t w e e n rats, w i t h some a n i m a l s s h o w i n g little or no response. W h i l e this c o u l d be interpreted to indicate that s o m e rats d o not find this procedure distressing, rats a l w a y s a v o i d e d CO2 concentrations o f a p p r o x i m a t e l y 1 5 % and higher d u r i n g a p p r o a c h - a v o i d a n c e testing. T h i s indicates that CO2 w a s l i k e l y aversive to a l l o f the rats d u r i n g CO2 euthanasia, but that the b e h a v i o u r a l responses that I m e a s u r e d are v a r i a b l e and l i k e l y p o o r measures o f distress d u r i n g f o r c e d gas exposure. In particular, it is interesting that escape b e h a v i o u r s were not observed i n a l l a n i m a l s , because the results o f m y thesis and that  of  K i r k d e n et a l . (2005) suggest that rats are h i g h l y m o t i v a t e d to a v o i d CO2. It is p o s s i b l e that some rats d i d not p e r c e i v e a potential to exit the exposure c h a m b e r and therefore d i d not try to 123  escape. W h i l e there is the p o s s i b i l i t y that rats w e r e p e r f o r m i n g relevant b e h a v i o u r s that I d i d not detect, the fact that s o m e rats r e m a i n e d c o m p l e t e l y m o t i o n l e s s throughout the exposure suggests this not to be the case. H o w e v e r , this raises the p o s s i b i l i t y that i n d i v i d u a l rats were c o p i n g w i t h the f o r c e d exposure i n different w a y s . I n d i v i d u a l s c a n r e s p o n d either p r o a c t i v e l y or r e a c t i v e l y w h e n c o n f r o n t e d w i t h a stressor ( r e v i e w e d b y K o o l h a s et a l . , 1999). H e n c e , another e x p l a n a t i o n for the v a r i a b i l i t y i n m y results is that rats were c o p i n g i n different m a n n e r s , w i t h s o m e s h o w i n g f r e e z i n g and others s h o w i n g escape b e h a v i o u r s . In  experiments  where  I  e x a m i n e d the b e h a v i o u r a l  responses o f rats d u r i n g  CO2  euthanasia, a n i m a l s were tested i n a n o v e l e n v i r o n m e n t . D u r i n g exposure to a predator o d o u r , rats have been f o u n d to s h o w behaviours  associated w i t h  fear w h e n tested i n a  novel  e n v i r o n m e n t , but not w h e n tested i n a f a m i l i a r e n v i r o n m e n t ( M o r r o w et a l . , 2 0 0 2 ) . T h i s f i n d i n g suggests that fear i n rats is enhanced b y a n o v e l e n v i r o n m e n t . It is p o s s i b l e that I m a y have o b t a i n e d different results i f a n i m a l s were tested i n a f a m i l i a r e n v i r o n m e n t . H o w e v e r , rats were tested i n a f a m i l i a r e n v i r o n m e n t d u r i n g a p p r o a c h - a v o i d a n c e testing a n d s t i l l a v o i d e d  CO2  concentrations greater than 1 5 % , i n d i c a t i n g that the o v e r a l l c o n c l u s i o n s o f the thesis are v a l i d . T h e m a i n l i m i t a t i o n o f a p p r o a c h - a v o i d a n c e testing is that it p r o v i d e s i n f o r m a t i o n o n l y about the rats' i n i t i a l p e r c e p t i o n o f CO2, a n d does not address a n y effect that m i g h t o c c u r w i t h c o n t i n u e d exposure u n t i l loss o f consciousness. F r o m the t i m e - c o u r s e o f CO2 euthanasia w i t h a m e d i u m f l o w rate, it appears that there is a p e r i o d o f at least 4 5 s b e t w e e n the onset o f a v e r s i o n and loss o f c o n s c i o u s n e s s . Distress d u r i n g the entire procedure c o u l d be assessed b y c o m p a r i n g g r a d u a l - f i l l CO2 exposure u n t i l loss o f consciousness w i t h a k n o w n a v e r s i v e s t i m u l u s . O n e w a y to e x a m i n e this w o u l d be to c o m p a r e the w i l l i n g n e s s o f a n i m a l s to re-enter the  exposure  c h a m b e r after exposure to each o f the s t i m u l i . F o r e x a m p l e , J o n g m a n et a l . (2000) used this m e t h o d to c o m p a r e p i g s ' a v e r s i o n to CO2 and to a n electric s h o c k d e l i v e r e d from a p r o d , and f o u n d that p i g s w o u l d m o r e r e a d i l y re-enter the exposure c h a m b e r after exposure to 9 0 % CO2 124  t h a n after exposure to electric shock. A n o t h e r m e t h o d o f c o m p a r i n g t w o aversive s t i m u l i is to use a v o i d a n c e - a v o i d a n c e testing, w h e r e a n i m a l s m u s t choose b e t w e e n t w o aversive s t i m u l i . T h e s t i m u l u s that is c h o s e n i s a s s u m e d to be the less aversive o f the t w o . F o r e x a m p l e , R u s h e n (1986) used a Y - m a z e to c o m p a r e a v e r s i o n to different h a n d l i n g techniques i n sheep. A n o t h e r l i m i t a t i o n o f the e x p e r i m e n t a l d e s i g n that I u s e d f o r a p p r o a c h - a v o i d a n c e testing w a s that I h a d to habituate the rats to CO2 exposure p r i o r to testing. T h i s resulted i n rats tolerating s l i g h t l y h i g h e r CO2 concentrations than w o u l d be o b s e r v e d o n a n i n i t i a l exposure, as w o u l d o c c u r d u r i n g euthanasia. H o w e v e r , it w a s necessary to habituate the rats to a v o i d a drift i n response to C 0 throughout the experiment. M o r e o v e r , I w a s able to estimate the m a g n i t u d e 2  o f this effect i n C h a p t e r 5.  6.3 Future directions 6.3.1 Other rodent species T h e results o f m y thesis suggest that g r a d u a l - f i l l C 0 euthanasia causes distress i n rats, 2  but w e d o n o t k n o w whether this i s also true f o r other rodent species s u c h as m i c e . P r e v i o u s studies o n distress d u r i n g CO2 euthanasia i n m i c e have been i n c o n c l u s i v e ( A m b r o s e et a l . , 2 0 0 0 ; B l a c k s h a w et a l , 1 9 8 8 ; B r i t t , 1 9 8 6 ; I w a r s s o n a n d R e h b i n d e r , 1993). L i k e the rat studies, these studies suffer f r o m p r o b l e m s w i t h e x p e r i m e n t a l d e s i g n , i n c l u d i n g a l a c k o f appropriate sample sizes, c o n t r o l groups a n d a c c l i m a t i z a t i o n before gas exposure. L e a c h et a l . (2002) demonstrated that m i c e w i l l a v o i d CO2 d u r i n g a preference test, but G o d b e y (personal c o m m u n i c a t i o n ) f o u n d that m i c e that are p r o v i d e d w i t h a f o o d r e w a r d d u r i n g g r a d u a l - f i l l CO2 euthanasia w i l l lose c o n s c i o u s n e s s w h i l e eating. T h i s latter result suggests that m i c e m i g h t be less averse to CO2 t h a n rats, but w e l l c o n t r o l l e d studies are needed to determine i f this is the case.  125  6.3.2 Alternative euthanasia methods G r a d u a l - f i l l CO2 euthanasia appears to cause distress i n rats, suggesting that this m e t h o d s h o u l d b e r e p l a c e d w i t h other methods that are m o r e h u m a n e . H o w e v e r , f e w studies have e x a m i n e d whether the other methods that are currently a v a i l a b l e cause distress i n rats, a n d i f so, whether they are better o r w o r s e than CO2 euthanasia. O n e a n i m a l w e l f a r e benefit o f CO2 euthanasia i s that it i n v o l v e s m i n i m a l h a n d l i n g a n d restraint o f the a n i m a l s . A s i d e f r o m CO2 euthanasia, the t w o m a j o r classes o f gas euthanasia agents are inhalant anaesthetics a n d inert gases, s u c h as a r g o n a n d n i t r o g e n , w h i c h are used to d i s p l a c e O2 a n d cause severe h y p o x i a . Inhalant anaesthetics are n o t k n o w n to be p a i n f u l or to cause d y s p n e a , but d o have a pungent o d o u r that rats m a y find unpleasant. U s i n g s i m p l e preference testing, L e a c h et a l . (2002b) demonstrated that rats w i l l a v o i d exposure to inhalant anaesthetics before l o s i n g c o n s c i o u s n e s s , but further research is needed to e x a m i n e the strength o f this a v e r s i o n . A s d i s c u s s e d i n C h a p t e r 1, h y p o x i a c a n cause d y s p n e a i n h u m a n s , but does not appear to do so d u r i n g spontaneous breathing ( C a b l e , 2 0 0 3 ; M o o s a v i et a l . , 2 0 0 3 ) . F u r t h e r m o r e , p i g s a n d poultry  w i l l enter a c h a m b e r c o n t a i n i n g 9 0 % a r g o n i n air ( 2 % O2) to access f o o d a n d  c o n s p e c i f i c s , a n d w i l l r e m a i n f o r l o n g e n o u g h to lose c o n s c i o u s n e s s ( R a j & G r e g o r y , 1 9 9 5 ; R a j , 1 9 9 6 ; W e b s t e r & F l e t c h e r , 2 0 0 4 ) . T h i s suggests that it m i g h t b e p o s s i b l e to k i l l rats w i t h h y p o x i a w i t h o u t c a u s i n g distress. H o w e v e r , i n contrast to these results w i t h other species, Hornett and Haynes  (1984) e x a m i n e d euthanasia b y h y p o x i a w i t h a g r a d u a l l y  increasing  c o n c e n t r a t i o n o f n i t r o g e n a n d f o u n d that it caused " p a n i c " (p.99) i n rats. F u r t h e r m o r e , L e a c h et a l . (2002a) f o u n d that rats a v o i d 90%> argon i n air before l o s i n g c o n s c i o u s n e s s i n a s i m p l e preference test. In C h a p t e r 3 , 1 e x p a n d e d o n this w o r k be e x a m i n i n g rats' a v e r s i o n to 9 0 % argon i n a i r u s i n g a p p r o a c h - a v o i d a n c e testing, a n d f o u n d that they w o u l d f o r g o a palatable f o o d r e w a r d i n order to a v o i d argon exposure. I n fact, the m e d i a n latency to leave w i t h argon w a s 126  o n l y 3 s, w h i c h w a s s i m i l a r to that seen f o r 2 0 % CO2. A s a n inert gas, argon is not thought to h a v e a p e r c e p t i b l e s m e l l , so the m o s t l i k e l y e x p l a n a t i o n f o r this a v o i d a n c e is d y s p n e a . It i s not clear w h y rats behave d i f f e r e n t l y f r o m other species, but it is p o s s i b l e that they are m o r e sensitive to h y p o x i a as a result o f b u r r o w - d w e l l i n g adaptations. E n h a n c e d sensitivity to h y p o x i a a n d h y p e r c a p n i a w o u l d be u s e f u l i n f o s s o r i a l species f o r a v o i d a n c e o f gas irregularities that c a n o c c u r u n d e r g r o u n d . W h i l e Rattus norvegicus is not f o s s o r i a l , this trait m a y be c o n s e r v e d a m o n g rodents. V e n t i l a t i o n i n f o s s o r i a l species is a c t u a l l y k n o w n to be less responsive to h y p o x i a a n d h y p e r c a p n i a than i n n o n - f o s s o r i a l species, l i k e l y i n o r d e r to tolerate the elevated CO2 (1 to 9.5%) and reduced 0  2  (6 to 2 0 % ) concentrations that o c c u r i n c l o s e d b u r r o w s ( r e v i e w e d b y  T e n n e y and B o g g s , 1986). H o w e v e r , the causes and d y n a m i c s o f d y s p n e a are still p o o r l y u n d e r s t o o d , a n d it is p o s s i b l e that these species e x p e r i e n c e a sharp rise i n d y s p n e i c sensations at l e v e l s o f h y p o x i a a n d h y p e r c a p n i a that are dangerous to s u r v i v a l . C a r b o n m o n o x i d e gas is another potential alternative euthanasia agent for laboratory rodents. C a r b o n m o n o x i d e i s a n o d o u r l e s s a n d n o n - i r r i t a t i n g gas that causes death b y direct t o x i c effects o n c e l l s , a n d b y c o m p e t i n g for O2 b i n d i n g sites o n h a e m o g l o b i n and p r e v e n t i n g s u f f i c i e n t d e l i v e r y o f O2 to b o d y tissues ( r e v i e w e d b y K a o and N a n a g a s , 2 0 0 5 ) . It has been suggested that c a r b o n m o n o x i d e p o i s o n i n g causes death w i t h o u t distress ( C l o s e et a l . , 1996). H o w e v e r , humans suffering from carbon monoxide  p o i s o n i n g report s y m p t o m s s u c h as  headache, nausea, chest p a i n , d y s p n e a , and elevated heart rate and breathing rate,  which  suggests that it does h a v e the potential to cause distress ( r e v i e w e d b y K a o a n d N a n a g a s , 2 0 0 5 ) . C a r b o n m o n o x i d e has not been e x a m i n e d as a euthanasia agent for rodents. H o w e v e r , C h a l i f o u x and D a l l a i r e (1993) f o u n d that c a r b o n m o n o x i d e caused v o c a l i z a t i o n s a n d signs o f agitation i n s o m e dogs before loss o f c o n s c i o u s n e s s . O n e m a j o r p r o b l e m w i t h the use o f c a r b o n m o n o x i d e i n the laboratory is that it is an odourless, n o n - i r r i t a t i n g gas that is p o t e n t i a l l y dangerous to h u m a n  127  health, and its use w o u l d therefore require precautions to ensure h u m a n safety. Further research e x a m i n i n g the effects o f c a r b o n m o n o x i d e o n rodents is necessary. C o m m o n l y used n o n - g a s euthanasia methods for laboratory rats i n c l u d e the p h y s i c a l m e t h o d s , s u c h as decapitation a n d c e r v i c a l d i s l o c a t i o n , and injectable anaesthetics. If  done  p r o p e r l y , the p h y s i c a l methods are q u i c k , and the m a i n a n i m a l w e l f a r e c o n c e r n is w i t h h a n d l i n g and restraint. H o w e v e r , the C C A C  ranks p h y s i c a l methods as o n l y c o n d i t i o n a l l y acceptable  because c o n s i d e r a b l e a n i m a l p a i n c a n o c c u r i f these methods are p e r f o r m e d  improperly.  I n j e c t i o n o f anaesthetics i n rats also requires restraint and some p a i n . It appears that a l l o f the c o m m o n l y u s e d euthanasia m e t h o d s for laboratory rats i n v o l v e factors that m i g h t cause distress before loss o f c o n s c i o u s n e s s . W h i l e m y thesis suggests that CO2 exposure also causes distress I cannot say whether this distress is m o r e o r less severe than that w h i c h occurs w i t h these other procedures. O n l y one study to date has c o m p a r e d a v e r s i o n to CO2 i n rats against other gas euthanasia methods. L e a c h et a l . ( 2 0 0 2 a , b) f o u n d that rats w o u l d r e m a i n i n inhalant anaesthetics and argon f o r l o n g e r than they w o u l d r e m a i n i n C 0 , but they 2  a v o i d e d a l l o f the agents before loss o f c o n s c i o u s n e s s . T h i s result indicates that a l l o f the agents are aversive o n i n i t i a l exposure, and that further research is necessary to determine w h i c h agent causes u n c o n s c i o u s n e s s w i t h the least l e v e l o f distress. O n e next step w o u l d be to c o m p a r e rats' a v e r s i o n to these different gas euthanasia agents u s i n g the a v e r s i o n testing methods described above i n 6 . 3 . R a t s w o u l d be exposed to each agent u n t i l u n c o n s c i o u s n e s s and then r e m o v e d . T h e i r w i l l i n g n e s s to re-enter compartments associated w i t h each agent w o u l d then be c o m p a r e d so that the agents c a n be r a n k e d a c c o r d i n g to the l e v e l o f a v e r s i o n that they cause. T h e s e m e t h o d s c o u l d also be u s e d to e x a m i n e whether rats f i n d gas euthanasia agents or h a n d l i n g and i n j e c t i o n m o r e aversive. T h e agent or m e t h o d that caused the least a v e r s i o n w o u l d be assumed to cause the least distress before unconsciousness. H o w e v e r , w i t h this m e t h o d o l o g y  128  it is  a s s u m e d that rats r e m e m b e r the e x p o s u r e ; therefore, it i s important to first ensure that none o f the agents affects m e m o r y .  6.4 Conclusions O v e r the past 10 years, there has been i n c r e a s i n g c o n c e r n about the use o f C 0  2  as a  euthanasia agent, for laboratory rodents. T h e H u m a n e S o c i e t y o f the U n i t e d States has c a l l e d f o r a b a n o n the use o f CO2 f o r euthanasia o f c o n s c i o u s rodents based o n a n i m a l w e l f a r e concerns ( C o n l e e et a l . , 2 0 0 5 ) , a n d recent d e v e l o p m e n t s have seen regulatory agencies, s u c h as the E u r o p e a n F o o d Safety A u t h o r i t y , suggest that CO2 s h o u l d not be used as a sole euthanasia agent f o r c o n s c i o u s a n i m a l s ( E F S A , 2 0 0 5 ) . T h e results o f m y thesis suggest that g r a d u a l - f i l l CO2 euthanasia causes distress i n laboratory rats. I have demonstrated that rats p e r f o r m escape b e h a v i o u r s d u r i n g g r a d u a l - f i l l CO2 euthanasia, a n d that they are at least m o d e r a t e l y averse to CO2 concentrations b e l o w those necessary to cause loss o f c o n s c i o u s n e s s , regardless o f the d e l i v e r y m e t h o d . T h e s e results suggest that laboratory rats s h o u l d not be euthanized w i t h CO2. H o w e v e r , p r e v i o u s research indicates that rats also s h o w a v e r s i o n to other euthanasia agents, so it i s not clear that a n y o f the agents currently a v a i l a b l e c a n i n d u c e u n c o n s c i o u s n e s s w i t h o u t distress. Further research is needed to determine w h i c h euthanasia agents cause the least amount o f distress i n rats a n d other rodent species, a n d to i d e n t i f y alternative m e t h o d s that c a n cause death w i t h o u t distress. .  129  6.5 References A m b r o s e , N . , W a d h a m , J . , M o r t o n , D . , 2 0 0 0 . R e f i n e m e n t i n E u t h a n a s i a . In: B a l l s , M . , v a n Z e l l e r , A . M . , H a i d e r , M . E . ( E d s ) , Progress i n the R e d u c t i o n , R e f i n e m e n t and R e p l a c e m e n t o f A n i m a l Experimentation, Elsevier Science, A m s t e r d a m , pp. 1159-1169. A n t o n , F., E u c h n e r , I., H a n d w e r k e r , H . O . , 1 9 9 2 . P s y c h o p h y s i c a l e x a m i n a t i o n o f p a i n i n d u c e d b y d e f i n e d CO2 pulses a p p l i e d to the nasal m u c o s a . P a i n 4 9 , 5 3 - 6 0 . B a n z e t t , R . B . , 1996. D y n a m i c response characteristics o f C O ^ - i n d u c e s air hunger. R e s p . P h y s i o l . 105, 4 7 - 5 5 . B l a c k s h a w , J . K . , F e n w i c k , D . C . , Beattie, A . W . , A l l a n , D.J., 1988. The behaviour o f chickens, m i c e and rats d u r i n g euthanasia w i t h c h l o r o f o r m , c a r b o n d i o x i d e and ether. L a b . A n i m . 2 2 , 67-75. B r i t t , D . P., 1996. T h e humaneness o f c a r b o n d i o x i d e as an agent o f euthanasia for laboratory rodents. In: E u t h a n a s i a o f U n w a n t e d , Injured or D i s e a s e d A n i m a l s or for E d u c a t i o n a l or S c i e n t i f i c P u r p o s e s , U n i v e r s i t i e s F e d e r a t i o n for A n i m a l W e l f a r e , Potters B a r , p p . 1 9 - 3 1 . C a b l e , G . C , 2 0 0 3 . I n - f l i g h t h y p o x i a incident i n m i l i t a r y aircraft: causes a n d i m p l i c a t i o n s f o r t r a i n i n g . A v i a t . Space E n v i r o n . M e d . 7 4 , 1 6 9 - 1 7 2 . C h a l i f o u x , A . , D a l l a i r e , A . , 1 9 9 3 . P h y s i o l o g i c and b e h a v i o r a l e v a l u a t i o n o f CO2 euthanasia o f adult dogs. A m . J . V e t . R e s . 4 4 , 2 4 1 2 - 2 4 1 7 . C h e n , X . , G a l l a r , J . , P o z o , M . A . , B a e z a , M . , B e l m o n t e , C , 1995. CO2 s t i m u l a t i o n o f the cornea: a c o m p a r i s o n b e t w e e n h u m a n sensation and nerve activity i n p o l y m o d a l n o c i c e p t i v e afferents o f the cat. E u r . J . N e u r o s c i . 7, 1 1 5 4 - 1 1 6 3 . C l o s e , B . , B a n i s t e r , K . , B a u m a n s , V . , B e r n o t h , E., B r o m a g e , N . , B u n y a n , J . , Erhardt, W . , F l e c k n e l l , P., G r e g o r y , N . , H a c k b a r t h , H . , M o r t o n , D . , W a r w i c k , C , 1996. R e c o m m e n d a t i o n s for euthanasia o f e x p e r i m e n t a l a n i m a l s : Part 1. L a b . A n i m . 3 0 , 2 9 3 - 3 1 6 .  130  C o e n e n , A . M . , D r i n k e n b u r g , W . H . , H o e n d e r k e n , R., v a n L u i j t e l a a r , G . L . , 1 9 9 5 . C a r b o n d i o x i d e euthanasia i n rats: o x y g e n s u p p l e m e n t a t i o n m i n i m i z e s signs o f agitation a n d a s p h y x i a . L a b . A n i m . 29, 262-268. C o n l e e , K . M . , Stephens, M . L . , R o w a n , A . N . , K i n g , L . A . , 2 0 0 5 . C a r b o n d i o x i d e for euthanasia: c o n c e r n s r e g a r d i n g p a i n a n d distress, w i t h s p e c i a l reference to m i c e a n d rats. L a b . A n i m . 3 9 , 137-161. C o o p e r , J . , M a s o n , G . , R a j , M . , 1 9 9 8 . D e t e r m i n a t i o n o f the a v e r s i o n o f f a r m e d m i n k ( M u s t e l a v i s o n ) to c a r b o n d i o x i d e . V e t . R e c . 1 4 3 , 3 5 9 - 6 1 . C u n n i n g h a m , D . J . C . , R o b b i n s , P. A . , W o l f f , C . B . 1986. Integration o f respiratory response to changes i n a l v e o l a r p a r t i a l pressures o f CO2 and O2 and i n arterial p H . In: C h e r n i a k , N . S . , W i d d i c o m b e , J . G . (eds), H a n d b o o k o f P h y s i o l o g y , S e c t i o n 3 : T h e R e s p i r a t o r y S y s t e m , V o l u m e II: C o n t r o l o f B r e a t h i n g , Part 2 , A m e r i c a n P h y s i o l o g i c a l S o c i e t y , W a s h i n g t o n , D . C . , pp.475-528 D a n n e m a n , P . J . , S t e i n , S . , W a l s h a w , S . O . , 1997. H u m a n e and p r a c t i c a l i m p l i c a t i o n s o f u s i n g c a r b o n d i o x i d e m i x e d w i t h o x y g e n for anaesthesia or euthanasia o f rats. L a b . A n i m . S c i . 4 7 , 376-85. D e S i m o n e , J . A . , L y a l l , V . , H e c k , G . . , F e l d m a n , G . M . 2001 A c i d detection b y taste receptor cells. R e s p . P h y s i o l . 129, 2 3 1 - 2 4 5 . D r i p p s , R . D . , C o m r o e , J . H . , 1 9 4 7 . T h e respiratory a n d c i r c u l a t o r y response o f n o r m a l m a n to i n h a l a t i o n o f 7.6 and 10.4 per cent CO2 w i t h a c o m p a r i s o n o f the m a x i m a l v e n t i l a t i o n p r o d u c e d b y severe m u s c u l a r exercise, i n h a l a t i o n o f CO2 and m a x i m a l v o l u n t a r y h y p e r v e n t i l a t i o n . A m . J . P h y s i o l . 149, 4 3 - 5 1 . E F S A , 2 0 0 4 . O p i n i o n o f the S c i e n t i f i c P a n e l o n A n i m a l H e a l t h a n d W e l f a r e o n a question related to " A s p e c t s o f the b i o l o g y and w e l f a r e o f a n i m a l s used for e x p e r i m e n t a l and other s c i e n t i f i c p u r p o s e s " - A d o p t e d b y the A H A W P a n e l o n 14th N o v e m b e r 2 0 0 5 ( Q u e s t i o n N ° 131  E F S A - Q - 2 0 0 4 - 1 0 5 ) . T h e E F S A J o u r n a l 2 9 2 , 1-46 F e n g , Y . , S i m p s o n , T. L., 2 0 0 3 . N o c i c e p t i v e sensation a n d s e n s i t i v i t y e v o k e d f r o m h u m a n c o r n e a and c o n j u n c t i v a s t i m u l a t e d b y CO2. Invest. O p h t h . V i s . S c i . 4 4 , 5 2 9 - 5 3 2 . G e r r i t z e n , M . A . , L a m b o o i j , E., H i l l e b r a n d , S . J . W . , L a n h a a r , J . A . C . , Pieterse, C , 2 0 0 0 . B e h a v i o r a l responses o f broilers to different gaseous atmospheres. P o u l t r y S c i . 7 9 , 9 2 8 - 9 3 3 . H a c k b a r t h , H . , K u p p e r s , N . , B o h n e t , W . , 2 0 0 0 . E u t h a n a s i a o f rats w i t h c a r b o n d i o x i d e — a n i m a l w e l f a r e aspects. L a b . A n i m . 3 4 , 9 1 - 9 6 . H e w e t t , T . A . , K o v a c s , M . S . , A r t w o h l , J . E . , Bennett, B . T . , 1 9 9 3 . A c o m p a r i s o n o f euthanasia m e t h o d s i n rats, u s i n g c a r b o n d i o x i d e i n p r e - f i l l e d a n d f i x e d f l o w rate filled c h a m b e r s . L a b . A n i m . Sci. 43, 579-582. H i l l , L., F l a c k , M . , 1908. T h e effect o f excess o f c a r b o n d i o x i d e and o f w a n t o f o x y g e n u p o n the t  r e s p i r a t i o n and the c i r c u l a t i o n . J . P h y s i o l . 3 7 , 7 7 - 1 1 1 . H o r n e t t , T . D . , H a y n e s , A . R . , 1984. C o m p a r i s o n o f c a r b o n dioxide/air m i x t u r e a n d nitrogen/air m i x t u r e for the euthanasia o f rodents. D e s i g n o f a system for i n h a l a t i o n euthanasia. A n i m a l Technology 35, 93-99. I w a r s s o n , K . , R e h b i n d e r , C , 1 9 9 3 . A study o f different euthanasia t e c h n i q u e s i n g u i n e a p i g s , rats, and m i c e . A n i m a l response and p o s t m o r t e m f i n d i n g s . S c a n . J . L a b . A n i m . S c i . 2 0 , 1 9 1 205. J o n g m a n , E . C . , Barnett, J . L . , H e m s w o r t h , P H . , 2 0 0 0 . T h e aversivenss o f c a r b o n d i o x i d e s t u n n i n g i n p i g s a n d a c o m p a r i s o n o f the CO2 stunner crate vs. the V - r e s t r a i n e r . A p p l . A n i m . Behav. Sci. 67, 67-76. K a o , L . W . , Nanagas, K . A . , 2005. Carbon monoxide poisoning. M e d . C l i n . N . A m . 89, 11611194.  132  K i r d e n , R., N i e l . , L., W e a r y , D . M . , 2 0 0 5 . H o w a v e r s i v e is gradual f i l l c a r b o n d i o x i d e euthanasia for rats? 2 0 0 5 C A L A S / A C S A L S y m p o s i u m P r o c e e d i n g s , V a n c o u v e r , B . C . , C a n a d a , p. 31.  K o o l h a s , J . M . , K o r t e , S . M . , D e B o e r , S . F . , v a n der V e g t , B . J . , v a n R e e n e n , C . G . , H o p s t e r , H . , de J o n g , I.C., R u i s , M . A . W . , B l o k h u i s , H . J . , 1 9 9 9 . C o p i n g styles i n a n i m a l s : current status i n b e h a v i o r and s t r e s s - p h y s i o l o g y . N e u r o s c i . B i o b e h a v . R. 2 3 , 9 2 5 - 9 3 5 . L a g n e a u x , D . , 1 9 8 6 . V e n t i l a t o r y responses o f the rat to m i l d h y p e r c a p n i s t i m u l a t i o n before and after a l m i t r i n e b i s m e s y l a t e . R e s p . P h y s i o l . 6 5 , 3 7 9 - 3 8 8 . L e a c h , M . C . , B o w e l l , V . A . , A l l a n , T . A . , M o r t o n , D . B . 2 0 0 2 a . A v e r s i o n to gaseous euthanasia agents i n rats and m i c e . C o m p a r a t i v e M e d . 5 2 , 2 4 9 - 2 5 7 . L e a c h , M . C . , B o w e l l , V . A . , A l l a n , T.F. & M o r t o n , D . B . 2002b. Degrees o f aversion shown by rats and m i c e to different concentrations o f i n h a l a t i o n a l anaesthetics. V e t . R e c . 1 5 0 , SOSSIS. L i o t t i , M . , B r a n n a n , S . , E g a n , G . , S h a d e , R , M a d d e n , L., A b p l a n a l p , B . , R o b i l l a r d , R., L a n c a s t e r , J . , Z a m a r r i p a , F . E . , F o x , P.T., D e n t o n , D . , 2 0 0 1 . B r a i n responses associated w i t h c o n s c i o u s n e s s o f breathlessness (air hunger). P r o c . N a t . A c a d . S c i . 9 8 , 2 0 3 5 - 2 0 4 0 . M o n t g o m e r y , K . C , 1 9 5 5 . T h e r e l a t i o n between fear i n d u c e d b y n o v e l s t i m u l a t i o n and exploratory behavior. J. C o m p . P h y s i o l . P s y c h o l . 4 8 , 254-260. M i n o w a , Y . , Ide, T., N i s h i n o , T., 2 0 0 2 . E f f e c t s o f f u r o s e m i d e o n C 0  2  ' ventilatory responsiveness  i n humans. P u l m . Pharmacol: Ther. 15, 363-368. M o o s a v i , S . H . , G o l e s t a n i a n , E., B i n k s , A . P . , L a n s i n g , R . W . , B r o w n , R., B a n z e t t , R . B . , 2 0 0 3 . H y p o x i c and h y p e r c a p n i c drives to breathe generate e q u i v a l e n t l e v e l s o f air hunger i n humans. J. A p p l . Physiol. 94, 141-154. M o r r o w , B . A . , Elsworth, J.D., R o t h , R . H . , 2002. Fear-like biochemical and behavioural responses i n rats to the predator odor, T M T , are dependent o n the exposure e n v i r o n m e n t . 133  Synapse 4 6 , 1 1 - 1 8 . N i s h i n o , T., Ide, T., S u d o , T., Sato, J . , 2 0 0 0 . Inhaled f u r o s e m i d e greatly alleciates the sensation o f experimentally induced dyspnea. A m . J. Respir. Crit. Care M e d . 161, 1963-1967. O n g , K . , K o r , A . , C h o n g , W . , Earnest, A . , W a n g , Y . , 2 0 0 4 . E f f e c t s o f i n h a l e d f u r o s e m i d e o n e x e r t i o n a l d y s p n e a i n c h r o n i c obstructive p u l m o n a r y disease. A m . J . R e s p i r . C r i t . C a r e M e d . 169, 1 0 2 8 - 1 0 3 3 . P e p p e l , P., A n t o n , F., 1 9 9 3 . R e s p o n s e s o f rat m e d u l l a r y d o r s a l h o r n neurons f o l l o w i n g intranasal n o x i o u s c h e m i c a l s t i m u l a t i o n : effects o f s t i m u l u s intensity, d u r a t i o n , and interstimulus interval. J. N e u r o p h y s i o l . 70, 2 2 6 0 - 2 2 7 5 . R a j , A . B . M . , 1996. A v e r s i v e reactions o f turkeys to argon, c a r b o n d i o x i d e and a m i x t u r e o f c a r b o n d i o x i d e and argon. V e t . R e c . 1 3 8 , 5 9 2 - 5 9 3 . R a j , A . B . M . , G r e g o r y , N . G . , 1 9 9 5 . W e l f a r e i m p l i c a t i o n s o f the gas stun n i n g o f pigs 1. D e t e r m i n a t i o n o f a v e r s i o n to i n i t i a l i n h a l a t i o n o f c a r b o n d i o x i d e or argon. A n i m . W e l f a r e 4, 273-280. R u s h e n , J . , 1986. A v e r s i o n o f sheep for h a n d l i n g treatments: p a i r e d - c h o i c e studies. A p p l . A n i m . B e h a v . S c i . 16, 3 6 3 - 3 7 0 . S h i m o y a m a , N . , S h i m o y a m a , M . , 2 0 0 2 . N e b u l i z e d f u r o s e m i d e as a n o v e l treatment for d y s p n e a i n t e r m i n a l cancer patients. J . P a i n S y m p t o m M a n a g . 2 3 , 7 3 - 7 6 . S m i t h , W . , H a r r a p , S . B . , 1997. B e h a v i o u r a l and c a r d i o v a s c u l a r responses o f rats to euthanasia u s i n g c a r b o n d i o x i d e gas. L a b . A n i m . 3 1 , 3 3 7 - 3 4 6 . S u d o , T., H a y a s h i , F., N i s h i n o , T . 2 0 0 0 . R e s p o n s e s o f t r a c h e o b r o n c h i a l receptors to i n h a l e d f u r o s e m i d e i n anesthetized rats. A m . J . R e s p i r . C r i t . C a r e M e d . 1 6 2 , 9 7 1 - 9 7 5 T e n n e y , S . M . , B o g g s , D . F . , 1986. C o m p a r a t i v e m a m m a l i a n respiratory c o n t r o l . In: C h e r n i a k , N . S . , W i d d i c o m b e , J . G . (eds), H a n d b o o k o f P h y s i o l o g y , S e c t i o n 3 : T h e R e s p i r a t o r y S y s t e m , V o l u m e II: C o n t r o l o f B r e a t h i n g , Part 2 , A m e r i c a n P h y s i o l o g i c a l S o c i e t y , W a s h i n g t o n , D . C . , 134  pp.475-528 T h u r a u f , N . , G u n t h e r , M . , P a u l i , E., K o b a l , G . , 2 0 0 2 . S e n s i t i v i t y o f the negative m u c o s a l potential to the t r i g e m i n a l target s t i m u l u s CO2. B r a i n R e s . 9 4 2 , 2 7 - 8 6 . W e b s t e r , A . B . , F l e t c h e r , D . L . , 2 0 0 4 . A s s e s s m e n t o f the a v e r s i o n o f hens to different gas atmospheres u s i n g an a p p r o a c h - a v o i d a n c e test. A p p l . A n i m . B e h a v . S c i . 8 8 , 2 7 5 - 2 8 7 .  135  

Cite

Citation Scheme:

    

Usage Statistics

Country Views Downloads
United States 11 1
France 7 0
China 6 12
Canada 4 0
Japan 3 0
United Kingdom 1 0
City Views Downloads
Unknown 9 4
Shenzhen 5 12
Ashburn 4 0
Tokyo 3 0
Vancouver 3 0
Wilmington 2 0
Lewes 1 0
San Francisco 1 0
Bloomington 1 0
Maple 1 0
Shanghai 1 0
Menlo Park 1 0

{[{ mDataHeader[type] }]} {[{ month[type] }]} {[{ tData[type] }]}
Download Stats

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0093022/manifest

Comment

Related Items