Flavor Chemistry of Animal Foods [1St Edition] 9780841204041, 9780841205178, 0-8412-0404-7

Table of contents :
Title Page......Page 1
Copyright......Page 2
ACS Symposium Series......Page 3
FOREWORD......Page 4
PdftkEmptyString......Page 0
PREFACE......Page 5
1 Progress in Animal Flavor Research......Page 7
Taste Stimuli and Responses to Them: Overview......Page 8
Taste Preferences: Comparisons Among Rats, Cats and Guinea Pigs......Page 10
Modification of Flavor Preferences by Individual Experience......Page 17
Interaction of Inherent Preferences and Effects of Experience......Page 19
Conclusions......Page 21
LITERATURE CITED......Page 22
2 Food Preference Behavior in Birds and Mammals......Page 27
Factors Influencing Food Preference Behavior......Page 29
Empirical Screening of Flavoring Agents, Enhancers, and Spices......Page 37
Intensifying Flavor with Extracted or Volatilized Compounds......Page 39
Conclusions......Page 43
Literature Cited......Page 44
3 Methodology of Behavioral Testing Associated with Development in Animal Foods......Page 49
Ingestional Method......Page 50
Instrumental Method......Page 58
Literature Cited......Page 69
Experimental......Page 72
Results and Discussion......Page 75
Literature Cited......Page 83
5 Bacterial Action and Chemical Signalling in the Red Fox (Vulpes vulpes) and Other Mammals......Page 84
Fermentative Scent Sources: I) Mammalian; the Anal Sac......Page 85
Fermentative Scent Sources: II) Environmental; Carrion......Page 89
Mammalian and Environmental Fermentative Scent Sources Compared......Page 90
Behavioral studies......Page 92
Literature Cited......Page 95
Taste......Page 98
Smell......Page 105
Literature Cited......Page 106
The Carnivores......Page 108
Neurophysiology of Fungiform Papillae Taste Systems......Page 110
Cat Fungiform Papillae Taste Systems......Page 112
Comparison of Cat and Dog Fungiform Papillae Taste Systems......Page 116
Flavor Chemistry......Page 123
Theoretical Considerations......Page 126
Human Comparisons......Page 128
Literature Cited......Page 132
Acknowledgment......Page 134
Nutrient Requirements......Page 135
Feed Ingredients......Page 137
Voluntary Feed Intake and Production......Page 138
Chemical Senses and Feeding......Page 142
Literature Cited......Page 145
The Development Process......Page 147
A. Search Stage......Page 148
B. Early Development......Page 149
0. Test Marketing......Page 154
10 Repellents to Protect Crops from Vertebrate Pests: Some Considerations for Their Use and Development......Page 156
The Chemical Defenses of Plants......Page 157
Poison Avoidance by Animals......Page 161
Applications of Conditioned Repellency......Page 162
Conclusion......Page 168
Literature Cited......Page 169
B......Page 172
C......Page 173
F......Page 174
H......Page 175
M......Page 176
P......Page 177
R......Page 178
S......Page 179
Z......Page 180

Citation preview

Flavor Chemistry of Animal Foods Roger W. Bullard, EDITOR U.S. Fish and Wildlife Service

A symposium sponsored by the Division of Agricultural and Food Chemistry at the 174th Meeting of the American Chemical Society Chicago, Ill., August 29, 1977

ACS SYMPOSIUM SERIES 67

AMERICAN

CHEMICAL

SOCIETY

WASHINGTON, D.C. 1978

In Flavor Chemistry of Animal Foods; Bullard, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

Library of CongressCIPData Flavor chemistry of animal foods. (ACS symposium series; 67 ISSN 0097-6156) Includes bibliographical references and index. 1. Feeds—Flavor and odor—Congresses. 2. Animals, Food habits of—Congresses. 3. Repellents—Congresses. I. Bullard, Roger W., 1937- . II. American Chemical Society. Division of Agricultural and Food Chemistry. III. Series: American Chemical Society. ACS symposium series; 67. SF97.7.F58 ISBN 0-8412-0404-7

664'.6 ACSMC 8

77-27295 67 1-175 (1978)

Copyright © 1978 American Chemical Society All Rights Reserved. The appearance of the code at the bottom of thefirstpage of each article in this volume indicates the copyright owner's consent that reprographic copies of the article may be made for personal or internal use or for the personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, Inc. for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to copying or transmission by any means—graphic or electronic—for any other purpose, such as for general distribution, for advertising or promotional purposes, for creating new collective works, for resale, or for information storage and retrieval systems. The citation of trade names and/or names of manufacturers in this publication is not to be construed as an endorsement or as approval by ACS of the commercial products or services referenced herein; nor should the mere reference herein to any drawing, specification, chemical process, or other data be regarded as a license or as a conveyance of any right or permission, to the holder, reader, or any other person or corporation, to manufacture, reproduce, use, or sell any patented invention or copyrighted work that may in any way be related thereto. PRINTED I N T H E U N I T E D STATES O F A M E R I C A

American Chemical Society Library 1155 16th St. N. W. InWashington, Flavor Chemistry Animal Foods; Bullard, R.; D.ofC. 20036 ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

ACS Symposium Series Robert F. Gould, Editor

Advisory Board Kenneth B. Bischoff

Nina I. McClelland

Donald G. Crosby Jeremiah P. Freeman

Joseph V. Rodricks

E. Desmond Goddard

F. Sherwood Rowland

Jack Halpern

Alan C. Sartorelli

Robert A. Hofstader

Raymond B. Seymour

James P. Lodge

Roy L. Whistler

John L. Margrave

Aaron Wold

In Flavor Chemistry of Animal Foods; Bullard, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

FOREWORD The ACS SYMPOSIUM SERIES was founded in 1974 to provide a medium for publishing symposia quickly in book form. The format of the SERIES parallels that of the continuing ADVANCES IN CHEMISTRY SERIES except that in order to save time the papers are not typeset but are reproduced as they are submitted by the authors in camera-ready form. As a further means of saving time, the papers are not edited or reviewed except by the symposium chairman, who becomes editor of the book. Papers published in the ACS SYMPOSIUM SERIES are original contributions not published elsewhere in whole or major part and include reports of research as well as reviews since symposia may embrace both types of presentation.

In Flavor Chemistry of Animal Foods; Bullard, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

PREFACE

Τhe vast accomplishments in human food technology during the past two decades have stimulated a more recent revolution in animal foods. As a result, higher quality, more palatable foods for domestic pets and food producing animals are being developed. As knowledge and exper­ tise expand, they are being applied to new problem areas. The most recent efforts have been with nondomestic animals. We are encroaching steadily ingly intensive management is required to prevent serious declines in animal populations. With our growing recreational needs for an expand­ ing human population, more wildlife resources are sought, and greater emphasis is being placed on wildlife refuges, game preserves, and zoos. Consequently, there is an escalating need to improve or supplement the food supplies of many diverse species. However, it is difficult to improve the quantity or quality of the food supply without affecting the behavior or physiology of wildlife. The solution will come through an integrated effort involving several scientific disciplines. In another area, we are finding better ways to protect our human food supply from vertebrate pests by using animal attractants and repel­ lents in management methods. Attractants entice animals to encounter items used in animal damage control or items used in census techniques to determine the population of problem species. Repellents are applied directly to plants or grains to protect them from birds or mammals. Flavor chemistry and food technology for animals is more complex than the mere adaptation of existing technology. Unlike humans, animals cannot appraise food or food additives directly. This problem requires an interdisciplinary research program that is structured far differently than those for human food research. For example, biologists and ecologists are often needed, and the behaviorist assumes a different and much expanded role. This volume presents a broad coverage of the subject, both from the standpoint of animal species and of the various scientific disciplines involved. Many different domestic and nondomestic animals are dis­ cussed by specialists in organic and analytical chemistry, biochemistry, behavior, biology, nutrition, and physiology. Together they provide a clearer understanding of the interrelationships among these various discivii In Flavor Chemistry of Animal Foods; Bullard, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

plines and how each contributes to thefieldofflavorchemistry of animal foods. S. Fish and Wildlife Service ROGER W. BULLARD Denver, CO

U.

October 25, 1977

viii

In Flavor Chemistry of Animal Foods; Bullard, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

1 Progress in Animal Flavor Research WILLIAM W. JACOBS, GARY K. BEAUCHAMP, and MORLEY R. KARE Monell Chemical Senses Center, University of Pennsylvania, 3500 Market St., Philadelphia, PA 19104

For a terrestrial animal, flavor may be defined as the composite sensation resulting from placing something in the mouth. Therefore, flavor may include geminal and other chemical perature and proprioceptive cues. Thus, the subjective sensation we call flavor is the result of interactions of a complex of receptors. The bulk of experimental work in this field has focussed upon one class of receptors and associated CNS processes, the taste system. There are powerful arguments that the taste system is a uniquely important component in regulating flavor perception and food intake (e.g., 1,2) , but other sensory components significantly influence flavor perception (e.g., 3, 4). Humans regard flavors as qualities which add to the aesthetics of dining. Applied flavor researchers devote most of their efforts toward making foods and beverages more acceptable to humans or domestic animals. However, flavor sensations are not confined to domestic species' analyses of food substances. Examples of non-food-related flavor perception can be found in the responses of male hamsters to female conspecifics' vaginal secretions and the responses of guinea pigs to conspecific and congeneric urines. These secretions are both sniffed and licked (5, 6), and there is evidence for both olfactory and vomeronasal involvement in the case of the hamster (7). Guinea pig urine appears to include a complex of active substances (8) and codes a great deal of information including species, sex, diet of donor and individual identity (5,9,10). The inputs of several chemical sense systems and hence perception of the flavor of the urine may be required for the processing of all of the information present. As yet, however, there is no experimental evidence that the taste system is crucial in the processing of information contained in mammalian secretions and excretions. Instead, taste is apparently rather specifically concerned with food and liquid intake. Consequently, taste responses are emphasized in this paper in a comparative-evolutionary approach toward flavor and its relation to food selection by wild and domestic species.

© 0-8412-0404-7/78/47-067-001$05.00/0

In Flavor Chemistry of Animal Foods; Bullard, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

2

FLAVOR

CHEMISTRY

OF ANIMAL

FOODS

Taste S t i m u l i and Responses t o Them: Overview The sensory r e c e p t i o n and response systems of organisms, l i k e t h e i r other f e a t u r e s , are shaped by s e l e c t i o n f o r c e s and are thus tuned t o meet the pressures of the a n c e s t r a l environment. Chemical d e t e c t i o n systems are found i n very p r i m i t i v e l i f e forms i n c l u d i n g b a c t e r i a , protozoans and c o e l e n t e r a t e s (11, 12, 13, 14). These species respond t o a v a r i e t y of s t i m u l i i n c l u d i n g some of those i d e n t i f i e d by humans as having one of the presumed (15) four b a s i c t a s t e s (sweet, s a l t y , sour and b i t t e r ) . I t i s s t r i k i n g that s i m i l a r c l a s s e s of substances are b e h a v i o r a l l y a c t i v e and that c e r t a i n types o f compounds e l i c i t r a t h e r s i m i l a r responses c r o s s phyletically. Some compounds i n the environment have had great i n f l u e n c e s upon s u r v i v a l of animal l i f e s i n c e i t f i r s t appeared. I t i s r e a sonable t o surmise tha these compounds would To evaluate t h i s p o s s i b i l i t y f o r compounds f a l l i n g i n t o the four c l a s s i c t a s t e q u a l i t i e s reported i n the human l i t e r a t u r e , a l i s t ing has been made of substances r e p o r t e d l y y i e l d i n g each of these q u a l i t i e s t o humans (Table I ) . Of the n a t u r a l l y o c c u r r i n g chemic a l s , i t can be seen t h a t many sugars, some D-amino a c i d s , as w e l l as L-alanine and g l y c i n e are i d e n t i f i e d as sweet. Such substances are found i n many foods. For example, f r e e sugars ( e s p e c i a l l y g l u cose, f r u c t o s e and sucrose) are present i n s u b s t a n t i a l amounts i n many f r u i t s and vegetables (20). The l i s t of s a l t y t a s t i n g substances contains numerous organi c and i n o r g a n i c s a l t s i n c l u d i n g some t o x i c substances (e.g., l i t h i u m c h l o r i d e ) , but a l s o some n u t r i t i o n a l l y important c a t i o n s and anions. Most abundant of a l l of these compounds i s sodium c h l o r i d e which, i n a d d i t i o n t o i t s p h y s i o l o g i c a l importance, was a l s o part of the medium (sea water) i n which many phyla evolved. For marine and t e r r e s t r i a l animals a l i k e , the most common s a l t y compound i s sodium c h l o r i d e w i t h the t o x i c ones much l e s s common. The d i s t r i b u t i o n of NaCl i s more patchy f o r t e r r e s t r i a l than f o r marine animals, however (21). Since NaCl i s an absolute r e q u i r e ment f o r complex organisms, i t i s not s u r p r i s i n g that n e a r l y a l l species t e s t e d respond t o i t . Sour t a s t i n g substances are a l l a c i d s (but not a l l a c i d s t a s t e sour) and are present i n many p o t e n t i a l food items, n o t a b l y f r u i t s . A c i d s , however, a l s o push the organism toward p h y s i o l o g i c a l a c i d o s i s under experimental c o n d i t i o n s and are not g e n e r a l l y p r e f e r r e d t o water a t any d e t e c t a b l e c o n c e n t r a t i o n . I n the l i q u i d medium i n which most phyla evolved, the a b i l i t y to detect a departure from normal environmental pH could a d a p t i v e l y t r i g g e r the avoidance of c o n d i t i o n s w i t h which many species are not p h y s i o l o g i c a l l y able t o cope. Nevertheless, there are a few s p e c i e s , the chicken f o r example (22), which t o l e r a t e and perhaps p r e f e r s o l u t i o n s of h i g h a c i d i t y .

In Flavor Chemistry of Animal Foods; Bullard, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

In Flavor Chemistry of Animal Foods; Bullard, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

- 16, 17, 18, 19.

Hydrochloric acid N i t r i c acid Acetic acid Butyric acid Succinic acid L a c t i c acid C i t r i c acid

Sodium c h l o r i d e Lithium chloride Ammonium c h l o r i d e Potassium c h l o r i d e Magnesium c h l o r i d e Sodium f l u o r i d e

Sucrose Glucose Fructose Raffinose Galactose Lactose Maltose Xylose Saccharin (Na) Cyclamate (Na) L-aspartyl-Lphenylalanine L-alanine D-histidine D-phenylalanine D-leucine D-tyrosine Glycine Beryllium chloride Thaumatin Monellin

References

Sour

Salty

Sweet Caffeine Nicotine Quinine compounds Strychnine Other a l k a l o i d s Urea Brucine Theobromine Naringin Glucoside Coumarins Terpene hydrocarbons Terpenoids L-leucine L-tryptophan L-tyrosine L-phenylalanine Magnesium s u l f a t e Denatorium benzoate (Bitrex) Sucrose o c t a a c e t a t e

Bitter

Table I . P a r t i a l l i s t i n g of compounds t a s t i n g sweet, s a l t y , sour o r b i t t e r to humans.

4

FLAVOR

CHEMISTRY

OF

ANIMAL

FOODS

Many n a t u r a l l y o c c u r r i n g b i t t e r t a s t i n g substances are t o x i c , i n c l u d i n g secondary compounds used as defenses by p l a n t s and a n i mals a g a i n s t depredation. Such substances, when detected, are g e n e r a l l y r e j e c t e d by organisms. I t i s not c l e a r i n every i n s t a n c e that r e j e c t i o n of b i t t e r substances i s independent of t h e i r nonsensory e f f e c t s , however (see l a t e r s e c t i o n s ) . T h i s b r i e f l i s t i n g of compounds f o r which humans r e p o r t one of the four b a s i c t a s t e sensations supports the n o t i o n that these compounds have great e c o l o g i c a l relevance f o r many s p e c i e s . This does not mean that each has equal relevance f o r a l l s p e c i e s , or that other s t i m u l i do not have relevance f o r some species ( c f . , 2^ 23). Indeed, w i t h i n one taxonomic group, the responses of i n d i v i d u a l species to v a r i o u s chemical s t i m u l i may d i f f e r c o n s i d e r a b l y . The degree to which these d i f f e r e n c e s represent adaptat i o n s to the unique e c o l o g i c a l c o n d i t i o n s encountered by each species has not been thoroughl Taste and food preferenc v a r i e t y of s p e c i e s , f o r a v a r i e t y of reasons and w i t h a v a r i e t y of procedures. The most f r e q u e n t l y used design has been that of R i c h t e r (24) which compares the acceptance of a t e s t s o l u t i o n w i t h that of water f o r a 24-hour p e r i o d . M o d i f i c a t i o n s of t h i s design i n c l u d e s h o r t e n i n g or lengthening the p e r i o d of exposure, changing the composition of the s o l v e n t and r e v e r s i n g the l o c a t i o n of t e s t and standard b o t t l e s i n an attempt to c o n t r o l f o r p o s i t i o n p r e f e r ences. An advantage of long t e s t d u r a t i o n s may be t h a t animals responses over periods of 24, 48, 72 or 96 hours are not subject to b r i e f whims of animals or to time-of-day e f f e c t s . Short t e s t proponents argue t h a t these f a c t o r s can be c o n t r o l l e d somewhat by c a r e f u l design and t h a t the r e s u l t s of s h o r t - d u r a t i o n t e s t s (10 min.- 6 hr.) are l e s s l i k e l y to be confounded by p o s t - i n g e s t i o n a l feedback e f f e c t s (e.g., 25). Some s t u d i e s have presented only one or two c o n c e n t r a t i o n s of a given s t i m u l u s . Others have t e s t e d whole s e r i e s , but may have presented concentrations i n ascending, descending or random o r d e r s , each of which may a f f e c t r e s u l t s d i f f e r e n t l y (26). Common procedures have seldom been used by d i f f e r e n t authors s t u d y i n g d i f f e r e n t s p e c i e s . Thus, c r o s s - s p e c i e s comparisons are d i f f i c u l t at best. The f o l l o w i n g d i s c u s s i o n w i l l center on three species for which data have been c o l l e c t e d on a v a r i e t y of s t i m u l i and under s e v e r a l d i f f e r e n t exposure times. 1

Taste Preferences: Comparisons Among Rats, Cats and Guinea P i g s Summaries of the t a s t e responses of l a b o r a t o r y r a t s (Rattus n o r v e g i c u s ) , domestic cats ( F e l i s c a t t u s ) and domestic guinea pigs (Cavia p o r c e l l u s ) appear i n Table I I . These forms represent three d i s t i n c t feeding types: h e r b i v o r e (guinea p i g ) , c a r n i v o r e (cat) and omnivore ( r a t ) . They are a l s o important research animals; thus there i s need to provide them w i t h acceptable d i e t s . F i n a l l y ,

In Flavor Chemistry of Animal Foods; Bullard, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

1.

Animal

JACOBS E T A L .

Flavor

Research

5

data are a l s o a v a i l a b l e on t a s t e responses of w i l d r e l a t i v e s o f each o f these s p e c i e s , p r o v i d i n g the o p p o r t u n i t y t o draw a few cautious i n f e r e n c e s concerning the e v o l u t i o n and l a b i l i t y of t a s t e preferences. Sweet Substances. Rats (Table I I ) show preferences f o r many substances t a s t i n g sweet t o humans. ( H e r e a f t e r , such responses w i l l be c a l l e d "sweet preferences" f o r the sake o f b r e v i t y ) . Cav i e s show preference f o r the only sugar (glucose) w i t h which they have been t e s t e d but do not p r e f e r i t a t as h i g h c o n c e n t r a t i o n s as do r a t s . Cats, on the other hand, e i t h e r are i n d i f f e r e n t t o o r r e j e c t sugars under most c o n d i t i o n s o f t e s t i n g ( c f . , d i s c u s s i o n i n 29). This i n d i f f e r e n c e t o sucrose and other sugars makes t h e cat (along w i t h some other s p e c i e s , _2, 41) an exception t o t h e g e n e r a l i t y o f sweet preferences among mammals (42) and i s i n accord w i t h e l e c t r o p h y s i o l o g i c a few f i b e r s s e n s i t i v e t clude, t h e r e f o r e , that a sweet t a s t e i s , f o r a l l p r a c t i c a l purposes, i n e r t as an a p p e t i t i v e cue f o r domestic c a t s , whereas r a t s and guinea p i g s can use t h i s cue i n food s e l e c t i o n . The responses to sweets by the domestic forms are very s i m i l a r t o those o f t h e i r w i l d r e l a t i v e s . M a i l e r (46, 47) found very s i m i l a r acceptance percentages (percent o f t o t a l f l u i d i n t a k e that i s t e s t s o l u t i o n i n s o l u t i o n vs. water choice t e s t ) o f s i n g l e conc e n t r a t i o n s of a v a r i e t y o f sugars by w i l d and l a b o r a t o r y Norway r a t s (Rattus n o r v e g i c u s ) . Laboratory r a t s increased f l u i d consumption g r e a t l y during these t e s t s whereas the w i l d type d i d not. Shumake, e t a l (48) found w i l d and l a b o r a t o r y r a t s t o be very simi l a r i n acceptance o f s o l i d d i e t s t r e a t e d w i t h sucrose i n a mechanized design i n which consumption d i f f e r e n c e s appear t o have been " c o n t r o l l e d out." Therefore, the d o m e s t i c a t i o n , o r more p r o p e r l y l a b o r a t o r i z a t i o n , o f Rattus has not a l t e r e d the sweet preferences of t h i s species but has a l t e r e d consumption, a r e l a t e d response. Wild c a v i e s showed higher acceptance and a broader preference range f o r glucose than d i d t h e i r domestic counterparts (33). The domestic c a v i e s acceptances became l i k e the responses o f the w i l d type (which remained c o n s i s t e n t ) upon r e t e s t i n g (34). Between-type consumption d i f f e r e n c e s a l s o were found, p a r t i c u l a r l y d u r i n g the a n i m a l s i n i t i a l exposures t o glucose. However, u n l i k e the s i t u a t i o n w i t h r a t s , i t was the w i l d c a v i e s who showed the g r e a t e r increases i n consumption o f sweet s o l u t i o n . These d i f f e r e n c e s between types a l s o diminished w i t h repeated t e s t i n g . These and other data i n d i c a t e that the w i l d cavy o r i e n t s more q u i c k l y t o s o l u t i o n cues than does the domestic form but f a i l t o provide any evidence f o r sensory d i f f e r e n c e s between them (34). Beauchamp, e t a l (29) have r e c e n t l y t e s t e d t a s t e responses o f twelve w i l d c a t s o f Genus Panthera ( l i o n s , P. l e o ; t i g e r s , _P. t i g r i s ; leopards, P. pardus; j a g u a r s , P. onca) i n short-term preference t e s t s . The r e s u l t s were not s u b s t a n t i a l l y d i f f e r e n t from 1

1

In Flavor Chemistry of Animal Foods; Bullard, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

In Flavor Chemistry of Animal Foods; Bullard, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

C i t r i c acid

Sour

26

f

2 6

27

Rej(.10*-.15M )

t

1

36

35

Pref (.008*-.05M *) Pref(.0092-.052M) 24

Pref(.0067-.61M) Rej(>1.05M)31

Pref(.006*-lM )

f

Pref (.22-1.39M )

31

26

Pref (.055-1.4M)

Pref(.055-1.6M)

Pref(.006*-1.6M) Pref(.011-.9M) Rej(>1.6M)27

Laboratory Rat

2 9

f

Rej(.06*-.48M )

37

Indif(.015*-.12M ) Rej(^.5M) 28

f

37

P r e f ( . l M ) Rej(.5M)28

1

Rej (.60M )

I n d i f 29

I n d i f 29

2 8

lndif > e

Domestic Cat

34

34

Rej(>.031M) 34

Rej(>_.5M)

Pref(.008-.25M) 34

f

3 2 , 3 3

Pref ( . l - . 4 M )

Pref(.2M o n l y )

Domestic Guinea P i g

Summary of responses of three f a m i l i a r mammals to some n a t u r a l sweet, s a l t y , sour and b i t t e r s t i m u l i i n s o l u t i o n v s . water choice t e s t s .

Sodium c h l o r i d e

Salty

Galactose

Maltose

Fructose

Glucose

Sucrose

Sweet

Table I I .

In Flavor Chemistry of Animal Foods; Bullard, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978. 3 5

38

y

Rej(>.000021M)

Rej(>.000275M)

1

Rej (.000008M* ')

Laboratory Rat

37

Rej (1.000005M*)

Rej (1.00025M*)

Domestic Cat

28

+

Rej(>.002M)

40

I n d i f (. 00016*-.00124M )

Domestic Guinea P i g

3

4

@ A preference f o r .25-.375M sucrose d i s s o l v e d i n .03M NaCl has been reported f o r c a t s . 29 subsequent study f a i l e d to f i n d such a preference.

t Highest concentrations used.

* I n d i c a t e s t h a t these c o n c e n t r a t i o n s were the lowest used i n the study c i t e d ,

5) Exposure times v a r i e d between s t u d i e s c i t e d .

30 A

Assignment of terms i n the l a t t e r case was based upon our experiences w i t h

conducting t h i s s o r t of t e s t i n g .

were l a c k i n g .

i n the c i t e d r e f e r e n c e s or by v i s u a l e v a l u a t i o n of t a b l e s and f i g u r e s i f s t a t i s t i c a l data

4) P r e f e r e n c e , I n d i f f e r e n c e and R e j e c t i o n were determined e i t h e r from s t a t i s t i c a l t e s t s presented

3) Rej = R e j e c t i o n

2) I n d i f = I n d i f f e r e n c e

1) P r e f = Preference

NOTES:

Quinine h y d r o c h l o r i d e

Quinine s u l f a t e

Bitter

Table I I . (continued)

8

FLAVOR

CHEMISTRY

OF

ANIMAL

FOODS

those w i t h domestic c a t s : Panthera p r e f e r r e d no sweet substances. S a l t y Substances. A l l three domestic species show a p r e f e r ­ ence f o r at l e a s t one hypotonic ( 102, 103, 104) and food s e l e c t i o n ( 2 ) . For e x t e r n a l and i n t e r n a l reasons, animals c o n t i n u a l l y e x e r c i s e t h e i r food s e l e c tion faculties. e

Conclusions This paper has s t r e s s e d f a c t o r s i n v o l v e d i n the e x h i b i t i o n of preferences and aversions by animals to p o t e n t i a l n u t r i e n t s . Perhaps the b i g g e s t advance i n animal f l a v o r research over the past f o r t y years has been the i d e n t i f i c a t i o n and experimental i s o l a t i o n of these f a c t o r s : inherent preferences, c o n d i t i o n e d a v e r s i o n s , neophobia and n e o p h i l i a . While each o f these f a c t o r s has been shown t o a f f e c t c h o i c e s , the manner i n which they i n t e r act f o r a given species must be determined e x p e r i m e n t a l l y i n each case. The i d e n t i f i c a t i o n of these f a c t o r s , however, provides the t o o l s needed to answer a p p l i e d questions concerning the feeding of p e t s , l i v e s t o c k and zoo animals and the c o n t r o l o f animal damage. I n doing such r e s e a r c h , the s u b j e c t s ' phylogenetic and ontogenetic h i s t o r i e s must always be considered.

In Flavor Chemistry of Animal Foods; Bullard, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

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FLAVOR CHEMISTRY OF ANIMAL FOODS

LITERATURE CITED 1. Hankins, W.G., Rusiniak, K.W. and Garcia, J., Behavioral Biology (1976) 18, 345-358. 2. Kare, M.R. and Beauchamp, G.K., In: Swenson, M.J. (ed.) "Dukes' Physiology of Domestic Animals," 713-730, Cornell University Press, Ithaca, N.Y., 1977. 3. Mozell, Μ., Smith, Β., Smith, P., Sullivan, R. and Swender, P., Archives of Otolaryngology (1969) 90, 367-373. 4. Mugford, R.A., In: Kare, M.R. and Maller, O. (eds.) "Chemical Senses and Nutrition, Vol. II," Academic Press, New York, in press. 5. Beauchamp, G.K., Physiology and Behavior (1973) 10, 589-594. 6. Johnston, R.E. and Zahorik, D.M., Science (1975) 189, 893-894. 7. Powers, J.B. and Winans, S.S., Science (1975) 187, 961-963. 8. Beruter, J., Beauchamp ical and Biophysica 264-271. 9. Beauchamp, G.K., Nature (1976) 263, 587-588. 10. Beauchamp, G.K., unpublished data. 11. Adler, J., In: Carlile, M.J. (ed.), "Primitive Sensory and Communication Systems," 91-100, Academic Press, London, 1975. 12. Beidler, L.M., In: Denton, D.A. and Coughlan, J.P. (eds.), "Olfaction and Taste: 5th International Symposium," 7176, Academic Press, New York, 1975. 13. Garcia, J . and Hankins, W.G., In: Denton, D.A. and Coughlan, J.P. (eds.) "Olfaction and Taste: 5th International Symposium," 39-45, Academic Press, New York, 1975. 14. Hodgson, E.S., In: Kare, M.R. and Mailer, O. (eds.) "The Chemical Senses and Nutrition," 7-18, Johns Hopkins Press, Baltimore, 1967. 15. McBurney, D., Chemical Senses and Flavors (1974) 1, 17-28. 16. Brand, J . G . , Nairn, M. and Kare, M.R., In: Rechcigl, M. (ed.) "Handbook Series in Nutrition and Food," CRC Press, Cleveland, in press. 17. Furia, T.E. , In: Furia, T.E. and Bellanca, N. (eds.) "Fenaroli's Handbook of Flavor Ingredients, Vol. I . , " 8-11, CRC Press, Cleveland, 1975. 18. Moskowitz, H . , In: Sipple, H.L. and McNutt, K.W. (eds.), "Sugars in Nutrition," 37-64, Academic Press, New York, 1974. 19. Solms, J., Journal of Agricultural and Food Chemistry (1969) 17_, 686-688. 20. Shallenberger, R.S., In: Sipple, H.L. and McNutt, K.W. (eds.), "Sugars in Nutrition," 67-80, Academic Press, New York, 1974. 21. Rozin, P., In: Hinde, R.A., Shaw, E. and Beer, C. (eds.) "Advances in the Study of Behavior, Vol. 6," 21-75 Academic Press, New York, 1976.

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22. Fuerst, W.F., Jr. and Kare, M.R., Poultry Science (1962) 41, 71-77. 23. Kare, M.R., In: Kare, M.R. and Halpern, B.P. (eds.) "Physio­ logical and Behavioral Aspects of Taste," 6-15, University of Chicago Press, Chicago, 1961. 24. Richter, C.P., Endocrinology (1939) 24, 367-371. 25. Cagan, R.H. and Mailer, O., Journal of Comparative and Physio­ logical Psychology (1974) 87, 47-55. 26. Kare, M.R. and Ficken, M.S., In: Zotterman, Y. (ed.) "Olfac­ tion and Taste," 285-297, Pergamon Press, Oxford, 1963. 27. Richter, C.P. and Campbell, K.H., American Journal of Physio­ logy (1940) 128, 291-297. 28. Carpenter, J.Α., Journal of Comparative and Physiological Psychology (1956) 49, 139-144. 29. Beauchamp, G.K., Mailer, O. and Rogers, J.G., Journal of Com­ parative and Physiologica 30. Bartoshuk, L.M., Harned 171, 699-701. 31. Richter, C.P. and Campbell, K.H., Journal of Nutrition (1940) 20, 31-46. 32. Bauer, F.S., Physiology and Behavior (1971) 6, 75-76. 33. Jacobs, W.W. and Beauchamp, G.K., Physiology and Behavior (1977) 18, 491-493. 34. Jacobs, W.W., in preparation. 35. Bernard, R.A., Halpern, B.P. and Kare, M.R,, Proceedings of the Society of Experimental Biology (N.Y.) (1961) 108, 784-786. 36. Harriman, A.E., American Midlands Naturalist (1968) 79, 396401. 37. Beauchamp, G.K. and Mailer, O., unpublished data. 38. Benjamin, R.M., Journal of Comparative and Physiological Psychology (1955) 48, 119-122. 39. Young, P.T., Burright, R.G. and Tromater, L.J., American Journal of Psychology (1963) 76, 205-217. 40. Warren, R. and Pfaffmann, C., Journal of Comparative and Physiological Psychology (1959) 52, 263-266. 41. Kare, M.R., In: Beidler, L.M. (ed.) "Handbook of Sensory Physiology: Chemical Senses Part 2: Taste," 277-292, Springer-Verlag, Berlin, 1971. 42. Pfaffmann, C. American Scientist (1964) 52, 187-206. 43. Boudreau, J.C., Bradley, B.E., Bierer, P.R , Kruger, S. and Tsuchitani, C., Experimental Brain Research (1971) 13, 461-488. 44. Cohen, M.J., Hagiwara, S. and Zotterman, Y . , Acta Physiologica Scandinavia (1955) 33, 316-332. 45. Pfaffmann, C., Journal of Neurophysiology (1955) 18, 429-440. 46. Mailer, O., In: Kare, M.R. and Mailer, O. (eds.) "The Chemical Senses and Nutritition," 201-212, Johns Hopkins Press, Baltimore, 1967. 47. Mailer, O. and Kare, M.R., Proceedings of the Society for r

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Experimental Biology and Medicine (1965) 119, 199-203. 48. Shumake, S.A., Thompson, R.D. and Caudill, C.J., Journal of Comparative and Physiological Psychology (1971) 77, 489494. 49. Abraham, S.F., Blaine, E.H., Denton, D.A., McKinley, M.J., Nelson, J.F., Shulkes, Α., Weisinger, R.S. and Whipp, G.T., In: Denton, D.A. and Coughlan, J.P. (eds.), "Olfaction and Taste: 5th International Symposium," 253-260, Academic Press, New York, 1975. 50. Bloom, J.C., Rogers, J.G. and Mailer, O., Physiology and Behavior (1973) 11, 95-98. 51. Goatcher, W.D. and Church, D.C., Journal of Animal Science (1970) 30, 777-783. 52. Goatcher, W.D. and Church, D.C., Journal of Animal Science (1970) 30, 784-790. 53. Mailer, O. and Kare M.R. Animal Behavio (1967) 15, 8-10 54. Jacobs, W.W., "Socia The Male-Female Association. Unpublished doctoral dissertation, University of Chicago, 1973. 55. Laughlin, M.E., Donovick, P.J. and Burright, R.G., Physiology and Behavior (1975) 15, 185-189. 56. Goatcher, W.D. and Church, D.C., Journal of Animal Science (1970) 31, 364-372. 57. Goatcher, W.D. and Church, D.C., Journal of Animal Science (1970) 31, 373-382. 58. Fisher, G.L., Pfaffmann, C. and Brown, E., Science (1965) 150, 506-507. 59. Valenstein, E.S., Kakolewski, J.W. and Cox, V.C., Science (1967) 156, 942-943. 60. Cote, J. and Brand, J.G., unpublished data. 61. Warren, R.P., Science (1963) 140, 808-809. 62. Warren, R.P. and Lewis, R.C., Nature (1970) 227, 77-78. 63. Chalupa, W., personal communication, 1977. 64. Hale, E.B., In: Hafez, E.S.E. (ed.), "The Behavior of Domestic Animals," Williams and Wilkins Co., Baltiomore, 1962. 65. Boice, R., Psychological Bulletin (1973) 80, 215-230. 66. White, T.D. and Boudreau, J.C., Physiological Psychology (1975) 3, 405-410. 67. Young, P.T., Psychological Review (1968) 75, 222-241. 68. Rogers, J.G., this symposium. 69. Harris, L.J., Clay, J., Hargreaves, F. and Ward, Α., Proceed­ ings of the Royal Society, Series Β (1933) 113, 161-190. 70. Richter, C.P., Harvey Lecture Series (1942-1943) 38, 63-103. 71. Rozin, P., Journal of Comparative and Physiological Psychology (1969) 69, 126-132. 72. Richter, C.P., In: Grasse, P.P. (ed.) "L'Instinct dans le Com­ portement des Animaux et de 1'Homme," Masson et Cie, Paris, 1956 73. Garcia, J., Hankins, W. and Rusiniak, Κ., Science (1974) 185, 824-831.

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74. Revusky, S.H., Psychonomic Science (1967)7,109-110. 75. Revusky, S.H., Journal of Comparative and Physiological Psychology (1968) 66, 777-779. 76. Beauchamp, G.Κ. and Mailer, O., In: Kare, M.R. and Mailer, O. (eds.) "Chemical Senses and Nutrition, Vol. II," Academic Press, New York, in press. 77. Hess,E.H.,"Imprinting," Van Nostrand Reinhold Co., New York, 1973. 78. Burghardt, G. and Hess, E.H., Science (1966) 151, 108-109. 79. Hess, E.H., Science (1964) 146, 1128-1139. 80. Dethier, V.G. and Goldrich, Ν., Science (1971) 173, 242-244. 81. Fuchs, J.L. and Burghardt, G.M., Learning and Motivation (1971) 2, 271-279. 82. Beauchamp, G.K., unpublished data. 83. Drickamer, L.C., Behaviour (1972) 41 269-287 84. Bronstein, P.M. an 18, 387-392. 85. Galef, Β.G., Journal of Comparative and Physiological Psy­ chology (1971) 75, 358-362. 86. Galef, Β.G. and Clark, M.M., Journal of Comparative and Physiological Psychology (1971) 75, 341-357. 87. Galef, Β.G. and Clark, M.M., Psychonomic Science (1971) 25, 15-16. 88. Galef, B.G. and Clark, M.M., Journal of Comparative and Physiological Psychology (1972)78,220-225. 89. Galef, B.G. and Henderson, P.W., Journal of Comparative and Physiological Psychology (1972)78,213-219. 90. Galef, B.G. and Sherry, D.F., Journal of Comparative and Physiological Psychology (1973) 83, 374-378. 91. Steiniger, F. von, Zeitschrift fur Tierpsychologie (1950) 7, 356-379. 92. Capretta, P.J., Petersick, J.T. and Stewart, D.J., Nature (1975) 254, 689-691. 93. Sclafani, Α., In: Novin, D., Wyrwicka, W. and Bray, G.A. (eds.) "Hunger: Basic Mechanisms and Clinical Implica­ tions," 281-295, Raven Press, New York, 1975. 94. Morrison, G.R., Journal of Comparative and Physiological Psychology (1974) 86, 56-61. 95. Desor, J.A., Mailer, O. and Turner, R.E., Journal of Com­ parative and Physiological Psychology (1973)84,496-501. 96. Young, P.T., Psychological Review (1966)73,59-86. 97. Nairn, M.. Kare, M.R. and Ingle, D.E., Journal of Nutrition, in press. 98. Braveman, N.S., Learning and Motivation (1974)5,182-194. 99. Braveman, N.S., Behavioral Biology (1975) 14, 189-199. 100. Wilcoxon, H.C., Dragoin, W.B. and Kral, P.Α., Science (1971) 171, 826-828. 101. Jacobs, W.W. unpublished data. 102. Michell, A.R., Physiology ad Behavior (1975) 14, 223-226.

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103. Wade, G.N. and Zucker, I., Journal of Comparative and Physiological Psychology (1969) 69, 291-300. 104. Zucker, I., Physiology and Behavior (1969) 4, 595-602. RECEIVED October 25, 1977.

In Flavor Chemistry of Animal Foods; Bullard, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

2 Food Preference Behavior in Birds and Mammals STEPHEN A. SHUMAKE U. S. Fish and Wildlife Service, Wildlife Research Center, Denver, CO 80225

The higher vertebrate and eloquent systems o searching, prey finding, sampling, and selection activities (1, 2). Some omnivorous species have a tremendous adaptive advantage in meeting their energy and nutritional requirements. For example, as one primary food source declines, the omnivores tend to sample new food sources or previously less palatable foods in order to survive (3, 4). Many migratory bird species including the African weaver finch or quelea (5) are able to survive and proliferate in spite of seasonal changes in the abundance of seeds and grains. The behavior and physiology of migratory birds appear to be directly correlated with environmental changes. For example, the quelea lay down fat stores before migrating from dry season areas (6). These birds also migrate with the rainy season to obtain enough food for themselves (seeds and grains) and for their young (green plant material and raw protein from termites and other insects). Certain herbivores such as antelope, mountain sheep, and elk are able to maintain themselves throughout the winter seasons of low food availability by moving to lower elevations (7) or by changing their microflora and microfauna to allow digestion of normally less nutritious plants such as locoweed or sagebrush. These observations point out the physiological and behavioral adaptiveness of wild birds and mammals and their ability to adopt new food habits. Partially for this reason, it is highly unlikely that any commercially available flavoring agents or flavor enhancers that have been developed for humans will have strong and durable flavor preference effects in wild species (8, 9). Instead, most wild animals appear to be adapted or conditioned to associate certain food odors, tastes, and flavors with beneficial effects (i.e., more energy, less nervousness, weight gain, and general health). Other flavors become associated with injurious sublethal poisoning (10) effects such as vomiting, diarrhea, or unconsciousness. Still, there are certain innate flavor preferences that exhibit themselves widely among the omnivores. For example, man, © 0-8412-0404-7/78/47-067-021$10.00/0 In Flavor Chemistry of Animal Foods; Bullard, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

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rodents, hogs, and most other mammals (VU 12) a l l show a preference for glucose, sucrose, and even non-nutritive saccharin that is independent of nutritional wisdom or experience. Bitter substances including quinine, sucrose octaacetate, and cyanide are avoided by birds (13), coyotes (14), rodents (1J5) , and herbivores (16). For most species this rejection behavior may be regarded as unlearned. The vast majority of food items consumed by omnivores contain extremely complex mixtures of flavors, and experience with the food is probably the single most important factor controlling preference behavior. Pure flavor sensation and animal preference for i t are extremely d i f f i c u l t to measure. P. T. Young's (V7) approach to this problem of measuring hedonic (pleasurable) or aversive (repellent) taste stimuli has been widely adopted and proposed as a standard method for evaluating taste preference in rats (18). Unfortunately, very few Past associations with tant than their sensory content (3, 19). Birds and mammals do not simply sample a new food item and then either continue or discontinue feeding on the basis of sensory (hedonic) content alone. Typically, the flavors or chemical components of the food items i n i t i a l l y act as signals for either nutritional or toxic effects. Later, palatability may actually be changed (20). In this respect, the chemical senses are similar to the visual or auditory senses. For example, i f gerbils are reared in their natural tunnel systems, they tend to quickly flee and hide from moving visual stimuli. If they are reared, like most laboratory rodents, in wire mesh cages with no opportunity to construct burrow systems, the gerbils show very l i t t l e avoidance of the same stimuli (21_). There are exceptions—if, for example, an animal is hormonally primed and presented with a "super normal" stimulus, the sheer intensity may be enough to evoke a genetically fixed response (22). Regardless of the wild species to be considered or the food flavor under study, the chemist involved in animal food flavor research and development should be aware of the numerous factors that ultimately influence and determine food preference behavior of birds and mammals. This review will not be an attempt to report a l l that is known about food preference behavior of birds and mammals. Such information could be of limited benefit to the chemist working on animal food flavor problems, but, as I will point out to the reader several times, each bird or mammal in a specific habitat is a somewhat unique entity. Laboratory data do not necessarily generalize to f i e l d situations and f i e l d experiments are often d i f f i cult i f not impossible to duplicate in the laboratory. Instead of an exhaustive review, I have elected to cover some important factors that can often influence flavor preference behavior. Next, a brief review is given of attempts to modify preference behavior by the empirical screening approach. Finally, an example of one approach to the problem of intensifying rice flavor to increase

In Flavor Chemistry of Animal Foods; Bullard, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

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SHUMAKE

Food

Preference

Behavior

23

rodent b a i t consumption i s covered i n some d e t a i l . A number o f suggestions i n regard to f u t u r e animal food f l a v o r research are then covered i n the conclusions s e c t i o n . Factors I n f l u e n c i n g

Food Preference Behavior

N u t r i t i o n a l F a c t o r s . Birds and mammals are e f f i c i e n t at food regulation. Even man, whose f l a v o r preference behavior i s l a r g e l y determined by s o c i a l , economic, and c u l t u r a l patterns (23) tends to g e n e r a l l y increase f a t intake i n c o l d environments, p r o t e i n content i n temperate zones, and carbohydrate content i n t r o p i c a l zones. Much of t h i s food s e l e c t i o n behavior, however, i s d i s t o r t e d by food a v a i l a b i l i t y whether i t be r e l a t e d to socioeconomic status or food crops p r e v a l e n t l y grown i n s p e c i f i c c l i m a t e s . From the standpoint o f animal h e a l t h n u t r i t i o n a l q u a l i t y o f foods ingested have a d i r e c d e f i n i t i o n , the f l a v o r q u a l i t i e meaning and can lead to n u t r i t i o n a l wisdom. As P. T . Young (24) and M. Kare (25) have often pointed o u t — t h e chemoreceptors stand as guarcls" to the alimentary c a n a l . But, j u s t how e f f i c i e n t are mammals and b i r d s at a c h i e v i n g n u t r i t i o n a l balance? The t a s t e or f l a v o r o f c e r t a i n e s s e n t i a l n u t r i e n t s such as glucose and sodium are immediately sought when a need a r i s e s i n mammals. Morrison and Young (26) and Nachman (27) b e l i e v e there i s an automatic increase i n the p a l a t a b i l i t y f o r s a l t s or sugars as they become d e f i c i e n t i n the d i e t . Some data (28) i n d i c a t e that there may be glucoreceptors i n the l i v e r that send neural information back to feeding and appetite centers i n the b r a i n . Mailer et a l . (29) have proposed a d i r e c t pathway to the b r a i n from the oral-pfiaryngeal area o f rats that regulates intake o f c e r t a i n n u t r i e n t s such as NaCl and glucose. The exact nature o f t h i s pathway and i t s p r o j e c t i o n area i n t o the lower b r a i n centers have not y e t been d e s c r i b e d . Pi 1 cher and Jarman (30) i n attempting to i n v e s t i g a t e t h i s o r i g i n a l f i n d i n g , b e l i e v e tFTat the hepatic system i s more important than the d i r e c t pathway hypothesis. Contreras & Hatton (31) have c i t e d some evidence that the sodium a p p e t i t e and p a l a t a b i l i t y change toward s a l t y t a s t i n g foods or l i q u i d s i n a l b i n o rats may be c o n t r o l l e d by the K/Na r a t i o i n t h e i r b l o o d . Morrison and Young (26) found that r a t s made sodium d e f i c i e n t by formalin i n j e c t i o n drank equal amounts o f e i t h e r 0.3 M NaCl or .03 M Na2CÛ3 s o l u t i o n even though NaCl contains f i v e times more sodium per u n i t volume. A g a i n , they conclude from these data that sodium d e p l e t i o n produces p a l a t a b i l i t y changes and that the amount o f " s a l t y " t a s t i n g s o l u t i o n drunk s i g n a l s s a t i e t y rather than sodium r e p l e t i o n . Thus, f o r some n u t r i e n t s , such as s a l t s or sugars, p a l a t a b i l i t y changes a u t o m a t i c a l l y regulate n u t r i t i o n a l balance i n r a t s . For thiamine (Vitamin B-|) d e f i c i e n c y , Rozin (32, 33) c i t e s r a t h e r convincing evidence t h a t the f l a v o r of the (fiet~Teading to vitamin B] s u f f i c i e n c y i s a s s o c i â t ! v e l y learned by r a t s . Food

In Flavor Chemistry of Animal Foods; Bullard, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

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odors may play a dominant r o l e i n vitamin enriched d i e t a r y s e l e c t i o n (3) or glucose i n j e c t i o n (34). T h i s same general f i n d i n g i s i m p l i c a t e d i n the e a r l y reports concerning s p e c i f i c a p p e t i t e f o r e s s e n t i a l vitamins (35, 36). For example, the vitamin d e f i c i e n t rats did not cue i n on the " f l a v o r " of vitamin B. Rather, they s e l e c t e d a new d i e t with a d i s t i n c t i v e (new) f l a v o r when the o l d f l a v o r e d d i e t l e d to continued d e f i c i e n c y symptoms. Diets cont a i n i n g calcium appear to a f f e c t rats i n a s i m i l a r manner (27). Although no s p e c i f i c appetites f o r vitamins A and D have been demonstrated (27), Bernard e t a l . (37) have shown t h a t vitamin A d e f i c i e n c y leads to decreased t a s t e s e n s i t i v i t y . Apparently, vitamin A i s e s s e n t i a l f o r normal t a s t e s e n s i t i v i t y o f the mammalian t a s t e buds (38) and both quinine r e j e c t i o n and NaCl s e l e c t i o n are retarded i n vitamin A d e f i c i e n t r a t s . There appear to be several d i f f e r e n c e s and some s i m i l a r i t i e s between t a s t e preferenc mals i n n u t r i t i o n a l d e f i c i e n c Hughes and Wood-Gush (39) were unable to demonstrate any evidence o f s p e c i f i c a p p e t i t e f o r sodium i n chickens fed e i t h e r a sodium d e f i c i e n t d i e t or given formalin i n j e c t i o n s . Young chicks were p a r t i c u l a r l y s u s c e p t i b l e to sodium c h l o r i d e t o x i c i t y . In r a t s , s p e c i f i c a p p e t i t e f o r sodium i s r e a d i l y e s t a b l i s h e d (40, 4 U 4 2 ) , probably because r a t s are more capable o f sodium e x c r e t i o n than most b i r d s , except f o r , perhaps c e r t a i n marine species t h a t possess s p e c i a l i z e d s a l t - s e c r e t i n g glands (43). Hughes and Wood-Gush (39) were able to e a s i l y demonstrate a s p e c i f i c a p p e t i t e f o r t h i a mine i n chickens s i m i l a r to that found i n rats (32). Thus, d i e t a r y d e f i c i e n c i e s can sometimes g r e a t l y i n f l u e n c e preference t e s t r e s u l t s in both birds and mammals. P h y s i o l o g i c a l Influences. Animals i n g e s t foods p r i m a r i l y to s a t i s f y energy requirements, metabolic needs, and f o r reproductive function. In s h o r t , food f l a v o r s become a s s o c i a t e d w i t h , or are c o r r e l a t e d w i t h , adequate d i e t under given environmental c o n d i tions. Animals t h a t do not s e l e c t adequate d i e t s do not s u r v i v e and reproduce. Thus, foods that c o r r e c t p h y s i o l o g i c a l d e f i c i e n c i e s or need s t a t e s are r e i n f o r c e d and food habits are then e s t a b l i s h e d (27). For example, the e f f e c t s o f i n f u s e d n u t r i e n t s i n t o the stomachs of r a t s have a marked i n f l u e n c e on feeding behavior. A d a i r , M i l l e r , and Booth (44) found that amino acids i n f l u s i o n produced an o v e r a l l decreased food consumption by a l b i n o r a t s . D-glucose alone, however, only s p e c i f i c a l l y suppressed consumption o f D-glucose s o l u t i o n s and not o v e r a l l consumption. Thus, the w i l l i n g n e s s o f an animal to eat any a v a i l a b l e food cannot be simply explained by a blood glucose-homeostasis mechanism ( i . e . , the glucostatic theory). Mixtures of c e r t a i n high c a l o r i e n u t r i t i v e and n o n - n u t r i t i v e sweeteners i n water s o l u t i o n can l e a d to excessive d r i n k i n g by l a b o r a t o r y rats (45). When o f f e r e d a mixture o f 3% glucose and 0.125 or 0.250% s a c c h a r i n i n d i s t i l l e d water, the rats consumed

In Flavor Chemistry of Animal Foods; Bullard, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

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f l u i d amounts approximately equal to t h e i r body weights every 24 hours. This excessive d r i n k i n g ( p o l y d i p s i a ) was explained as a s y n e r g i s t i c a c t i o n o f the mixture that l e d to a h i g h l y p a l a t a b l e sweet t a s t e without an a s s o c i a t e d high c a l o r i c c o n t e n t , while the s l i g h t l y b i t t e r t a s t e of s a c c h a r i n was masked by glucose. M a i l e r (46) has i n d i c a t e d that w i l d Norway rats (Rattus norvegicus) are c a l o r i c a l l y s e l e c t i v e i n t h e i r food h a b i t s . Thus, p a l a t a b i l i t y f a c t o r s could play a l e s s e r r o l e i n food s e l e c t i o n by w i l d rodents as compared with l a b o r a t o r y r a t s . I have found, however, t h a t w i l d Norway rats e x h i b i t p o l y d i p s i a when o f f e r e d the glucose and s a c c h a r i n mixture. A group o f s i x w i l d Norway r a t s drank an average o f 362 ± 21 ml o f f l u i d per day. Six w i l d r i c e f i e l d rats (Rattus r a t t u s mindanensis), on the other hand, d i d not show such extreme p o l y d i p s i c behavior, d r i n k i n g on the average 78 ± 8 ml of the f l u i d mixtur day Th f thi species d i f f e r e n c e was no combinations o f water, s a c c h a r i n s o l u t i o n , glucose s o l u t i o n , and the mixture. A p p a r e n t l y , both glucose and s a c c h a r i n and t h e i r i n t e r a c t i o n at e i t h e r the r e c e p t o r o r c a l o r i c r e g u l a t i n g l e v e l , c o n t r i b u t e to the species d i f f e r e n c e . As i n d i c a t e d above, w i l d rats do not always s e l e c t c a l o r i c a l l y balanced d i e t s , e s p e c i a l l y under l a b o r a t o r y c o n d i t i o n s . In attempting to f u r t h e r understand c a l o r i c r e g u l a t i n g and feeding systems o f r a t s , Piquard e t a l . (47) have found that a 25% d a i l y c a l o r i e glucose i n f u s i o n i n t o the r a t ' s c i r c u l a t o r y system v i a i n t r a c a r d i a c c a t h e t e r produced an 80% decrease i n intake o f g l u c o s e - t r e a t e d food. Amino a c i d and l i p i d i n f u s i o n produced decreases i n intake o f p r o t e i n s , l i p i d s , and g l u c i d s as w e l l . Booth and Campbell (48) found that i n s u l i n i n f u s i o n i n t o the c i r c u l a t o r y system of rats produced increased food consumption but they could not show that i n f u s i o n s o f f a t t y acids (the breakdown product of f a t s ) had any e f f e c t s on the food intake o f a l b i n o r a t s . Campbell and Davis (28) have shown t h a t both duodenal and portal glucose i n f l u s i o n s w i l l reduce l i c k i n g rates f o r glucose s o l u t i o n s . Thus, at l e a s t f o r glucose, sweet t a s t e p a l a t a b i l i t y and subsequent c a l o r i c r e g u l a t i o n may be c o n t r o l l e d to some degree by chemoreceptors i n the l i v e r . Other p h y s i o l o g i c a l v a r i a b l e s such as circadium rhythms (49), estrus c y c l e (50, 51), sexual hormones (52), and i n s u l i n l e v e l (53) can have i n f l u e n c e s on odor d e t e c t i o n and t a s t e preference or perception i n r a t s . A d e t a i l e d review o f these e f f e c t s i s beyond the scope of t h i s r e p o r t . However, f l a v o r researchers dealing with improving the food acceptance o f l i v e s t o c k , domestic p e t s , or w i l d animals should be aware that these v a r i a b l e s can a f f e c t f l a v o r preference t e s t r e s u l t s . Postingestional factors such as l e v e l of c i r c u l a t i n g blood glucose (54), bulk or f i b e r content o f the food ( 7 ) , and s u b l e t h a l i l l n e s s e f f e c t s (33) a l s o often play some r o l e i n preference t e s t r e s u l t s especially"when the animals are o f f e r e d continuous access or long-term access (>l-2 hrs) to the t e s t food or foods.

In Flavor Chemistry of Animal Foods; Bullard, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

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Genetic Factors and the E f f e c t s o f Domestication. Domestic a t i o n of the w i l d Norway r a t has l e d to c e r t a i n s u b t l e changes in f l a v o r preference behavior and the reactions o f d i f f e r e n t r a t s t r a i n s to new foods. M i t c h e l l , Beatty, and Cox ( 5 5 ) found that two d i f f e r e n t w i l d Norway r a t populations produced F] progeny that d i f f e r e d i n t h e i r i n i t i a l acceptance of a new food i n a new feeder. Norway r a t FT progeny from a pig farm a l s o showed a higher incidence o f p o i s o n - e l i c i t e d p i c a a f t e r cyclophosphamide i n j e c t i o n than d i d the progeny from Norway rats captured on a coastal i s l a n d . These d i f f e r e n c e s i n feeding behavior could be the product o f s e l e c t i o n pressure because rats i n farming areas would be much more l i k e l y to contact r o d e n t i c i d e s and traps than rats l i v i n g on an i s o l a t e d i s l a n d . Water, as a p a r t i a l physical b a r r i e r , could have a l s o l e d to more inbreeding among the i s l a n d r a t p o p u l a t i o n . In any event, the r e s u l t s tend to r e i n f o r c e the notion that l o n g i t u d i n a l studies are neede B r i e f - e x p o s u r e preferenc Kappauf ( 5 6 ) have not demonstrated genetic changes i n t a s t e p r e f erence b e f i â v i o r of Long-Evans hooded versus l a b o r a t o r y b r e d - a n d reared w i l d Norway rats ( 5 7 J . Thus, food f a m i l i a r i t y and r e a r i n g conditions may be, i n some i n s t a n c e s , more important determiners of t a s t e preference behavior and food acceptance than the more s u b t l e g e n e t i c e f f e c t s . Jackson ( 5 8 ) , f o r example, reported that roof rats i n c e n t r a l F l o r i d a feed e x t e n s i v e l y on oranges. Other Southern r o o f rats do not show much acceptance o f c i t r u s as a food item. In the P a c i f i c i s l a n d s , r o o f rats r e a d i l y take coconut on some i s l a n d s and ignore t h i s source o f food on other i s l a n d s . There i s , however, l i t t l e doubt that genetic i n f l u e n c e s can play a r o l e i n determining t a s t e preference behavior. For example, mice o f d i f f e r e n t species show d i f f e r e n t preference responses to 10% glucose versus 1 0 % f r u c t o s e ( 5 9 ) , d i f f e r e n t c o n d i t i o n e d a v e r sion responses to various sugars JEO), and d i f f e r e n t saccharin preference behavior ( 6 1 ) . Wenzel ( 6 2 ) cautions researchers that domesticated and l a b o ratory animals ( a l b i n o r a t s , pigeons, and chickens) may be much less s e n s i t i v e to t a s t e and odor s t i m u l i than are w i l d mammals and birds. For example, domestic chickens are able to compensate f o r reduced c a l o r i c content i n t h e i r d i e t by d r i n k i n g l a r g e r q u a n t i t i e s o f 10% sucrose i n water s o l u t i o n . Normally, however, chickens are i n d i f f e r e n t to sucrose. Kare and M a i l e r ( 6 3 ) found, on the other hand, t h a t Red Jungle fowl tend to p r e f e r sucrose s o l u t i o n s at a l l times and appear to be s u p e r i o r to the domestic chicken i n caloric regulation. Likewise, there i s some evidence ( 4 6 ) that c a l o r i c r e g u l a t i o n i n w i l d rodents i s u s u a l l y s u p e r i o r to t h a t demonstrated by l a b o r a t o r y r a t s . In terms o f animal food f l a v o r development as an a p p l i e d endeavor, these v a r i a b l e s o f domestication and genetic e f f e c t s do not g e n e r a l l y r e q u i r e separate analyses. The reported data do

In Flavor Chemistry of Animal Foods; Bullard, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

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imply, however, t h a t the f l a v o r research bioassay should i n c o r p o rate the t a r g e t s p e c i e s , and should d u p l i c a t e c e r t a i n aspects o f the natural ecosystem o f the t a r g e t mammal or b i r d . Behavioral and E c o l o g i c a l I n f l u e n c e s . Much o f the e c o l o g i c a l l i t e r a t u r e r e l a t e d to food h a b i t s , p r e f e r e n c e s , and aversions o f mammals deals with complex predator-prey r e l a t i o n s h i p s . Pearson (64) observed a c y c l i c a l r e l a t i o n s h i p between m i c r o t i ne rodent population l e v e l s and the abundance o f mammalian predators ( e . g . , skunks, foxes, weasels, and f e r a l c a t s ) . Because these predators r e l y on rodents as a primary food source, they tend to delay recovery o f lower rodent population c y c l e s . As rodent populations reach high d e n s i t y l e v e l s , d i s e a s e , p a r a s i t e s , and s t r e s s along with p r é d a t i o n pressure are thought to cause rodent population d e c l i n e s . When the m i c r o t i ne prey population has ebbed predators w i l l e i t h e r s t a r v e or emigrate Macdonald (65) ha red foxes (Vulpes vu 1 1 pes) i n great d e t a i l i n the f i e l d . The p a r t i c u l a r prey species taken can depend on several f a c t o r s such as prey a v a i l a b i l i t y and i t s a n t i p r e d a t o r b e h a v i o r , the r e l a t i v e energy costs o f hunting the prey, and the s i z e and n u t r i e n t value of the prey. Foxes took f i e l d mice i n preference to bank voles and wood mice. They a l s o tended to p r e f e r Microtus to Peromyscus. Moles and common shrews tended to be avoided as prey by the red fox. Fresh carcasses o f other carnivores ( e . g . , weasels, badgers, and foxes) were inspected by foxes but were not e a t e n . Instead, foxes would o f t e n mark t h e i r l o c a t i o n s with urine or f e c e s . The general pattern seemed to i n d i c a t e that foxes p r e f e r r e d the f l e s h o f herbivores and avoided the f l e s h o f i n s e c t - and meat-eating animals. The study could provide some clues to the food f l a v o r chemist as to p o s s i b l e s t a r t i n g points i n developing food f l a v o r s f o r f o x e s , dogs, or other c a n i d s . Apfelback (66) has observed that p r e y - c a t c h i n g behavior o f polecats i s highly" dependent on o l f a c t o r y cues. He found that polecats fed e i t h e r dead chicks o r dead rodents f o r t h e i r whole l i v e s showed no i n t e r e s t i n the odor o f the other prey and refused to eat i t . Searching behavior i s apparently only shown by p o l e cats when they contact the odor of the f a m i l i a r prey item. These demonstrated e f f e c t s , although p r i m a r i l y shown i n pen t e s t s , may i n d i c a t e a s e n s i t i v e period ( i . e . , through the fourth month o f l i f e ) during which c e r t a i n carnivores imprint on c e r t a i n prey items as food. With rodents, such o l f a c t o r y or food f l a v o r i m p r i n t i n g i s d i f f i c u l t to demonstrate under both l a b o r a t o r y and s e m i - f i e l d conditions. Some o f the e a r l i e r attempts (15^, 67^, 68) f a i l e d to show any e f f e c t s o f e a r l y feeding experience ( i . e . , post weaning) with l a b o r a t o r y rats or guinea p i g s . More recent reports (69, 70_, 71) have shown that temporary e f f e c t s can be demonstrated f o r food odor and food f l a v o r s i f rats are exposed to the s t i m u l i very

In Flavor Chemistry of Animal Foods; Bullard, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

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e a r l y i n l i f e ( i . e . , preweaning or during the f i r s t postnatal week of l i f e ) . Perhaps more important than p o s s i b l e f l a v o r i m p r i n t i n g e f f e c t s , r e s u l t i n g from the f i r s t foods eaten by newly weaned r a t s , are t h e i r maternal feeding experiences with s p e c i f i c f l a v o r s from l a c t a t i n g dams (72). Galef and Henderson (73) demonstrated that weanling rats would a c t i v e l y seek and p r e f e r e n t i a l l y feed on the d i e t fed to l a c t a t i n g dams during the nursing p e r i o d , even when the d i e t was normally l e s s p a l a t a b l e . Presumably, the f l a v o r o f the dam's d i e t w i l l a f f e c t the f l a v o r of her milk to some degree. Another important f a c t o r t h a t determines which food a r a t pup i s most l i k e l y to eat i s the feeding s i t e s e l e c t e d by parent rats (74). This appears to be a form of s o c i a l i m i t a t i o n that the pups d i s p l a y toward adults that are a c t i v e l y f e e d i n g . A t h i r d f a c t o r t h a t can i n f l u ence r a t pup feeding preference i s the existence o f a maternal pheromone (75, 76). Th the odor a n c T f l a v o r q u a l i t excreted f o l l o w i n g p a r t u r i t i o n , and r a t pups from such mothers tend to p r e f e r the odor and f l a v o r o f the mother's d i e t . Stimulus f a m i l i a r i t y (77) appears to be the most important aspect of these s o c i a l l y transmitted f l a v o r preference e f f e c t s . F i n a l l y , a fourth f a c t o r t h a t leads to feeding s i t e s e l e c t i o n by r a t pups (78) appears to be other o l f a c t o r y cues that r e s u l t from urine~cTe p o s i t s l e f t by a d u l t r a t s at s p e c i f i c feeding s i t e s . Flavors o f the most f a m i l i a r and normally p r e f e r r e d food items can, of course, determine which foods are most f r e q u e n t l y s e l e c t e d by the adult r a t s . R i c e f i e l d rats (R. r. mindanensis) show r i c e v a r i e t a l preference (79) and tend to p r e f e r Pal ay over Glutenous r i c e i n f i e l d rodent b a i t i n g programs. We have confirmed t h i s r i c e v a r i e t a l preference e f f e c t i n c l o s e d colony s e m i - f i e l d e n v i ronments with small groups o f a d u l t r i c e f i e l d r a t s . As i n d i c a t e d in Table I, the rats showed the highest consumption o f FK-178A r i c e (P < .01) and Milagrosa was p r e f e r r e d to the other two t e s t e d v a r i e t i e s , IR-20 and C-4 (P < .05). Under actual f i e l d c o n d i t i o n s , most rats would only contact one or two s i m i l a r v a r i e t i e s so t h a t these data do not allow f o r p r e d i c t i v e value i n f o r e c a s t i n g damage by the r a t s . However, the data do imply that s u b t l e changes i n the natural r i c e f l a v o r s of b a i t s could p o t e n t i a l l y i n f l u e n c e the extent o f b a i t acceptance. Some food h a b i t studies with stomach content or f e c a l matter analyses as measures take r e l a t i v e food a v a i l a b i l i t y {80, 81) i n t o account. Fellows and Sugihara (82), by t h i s method, were I F l e to determine t h a t Norway and Polynesian r a t s i n and near Hawaiian sugarcane f i e l d s showed high acceptance o f broad-leaved f r u i t s (melastoma, passion f r u i t , guava, thimble b e r r y , and popolo). The young (< 7 mo) of both r a t species showed avoidance o f grass veget a t i o n , whereas adults (7-24 mo) showed some passive acceptance. Such changes i n food acceptance could r e f l e c t e i t h e r ontogenetic ( e . g . , development o f a more adequate d i g e s t i v e system f o r e x p l o i t ing n u t r i e n t s from more food sources) or l e a r n i n g e f f e c t s ( e . g . ,

In Flavor Chemistry of Animal Foods; Bullard, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

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Behavior

a s s o c i a t i n g the n u t r i t i v e value o f grasses with t h e i r f l a v o r s ) . F i e l d i n v e s t i g a t i o n s that consider what i s being eaten i n r e l a t i o n to abundance can provide some of the most useful preference data f o r w i l d b i r d s and mammals. However, these s t u d i e s can be extremely time-consuming and undoubtedly r e q u i r e a great deal o f systematic assessment. Table I.

Day 1 2 3 4 5 6 7 8 9 10 11 12

Rice b a i t consumption (g) o f eight r a t c o l o n i e s four v a r i e t i e s o f r i c e (Mean ± S . E . ) .

Milagrosa 18.7 15.1 14.4 8.9 15.6 6.6 4.9 4.0 10.1 6.9 5.0 3.9

+ + + + + + + + + + ± +

4.8 4.2 3.3 1.5 5.2 2.3 2.1 1.4 3.9 2.7 2.6 0.9

Rice v a r i e t y FK-178A IR-20 26.4 + 5.7

38.7 37.4 49.9 49.1 49.4 23.5 36.6 34.9 36.6

+ + + + + + + + +

4.2 5.1 6.7 7.5 8.6 3.0 4.8 3.4 5.7

C-4

7.4 + 2.4

7.8 10.5 4.6 9.1 8.3 9.8 5.6 11.1 8.1

+ + + + + + + + ±

for

1.6 4.2 1.3 3.1 3.6 4.0 1 .3 3.0 1 .1

7.4 + 2.5

8.6 8.8 9.9 6.6 10.2 8.5 12.5 2.5 6.6

+ + + + + + + + +

1.4 1.4 3.2 2.2 3.3 2.0 5.5 1.0 2.5

Behavioral and e c o l o g i c a l e f f e c t s on f l a v o r acceptance or r e j e c t i o n i n b i r d s are more d i f f i c u l t to c h a r a c t e r i z e than those found in mammals. For example, Kare and Ficken (25) reviewed the e a r l y work concerned with t a s t e preference responses o f chickens with t r e a t e d versus untreated water over r e l a t i v e l y long exposure periods. I t was g e n e r a l l y found that chickens were i n d i f f e r e n t to a large number o f carbohydrates ( e . g . , s u c r o s e , glucose, l a c tose) except f o r xylose that was s t r o n g l y r e j e c t e d . Chickens a l s o c o n s i s t e n t l y r e j e c t e d c h l o r i d e s a l t s (ammonium, c a l c i u m , and f e r r i c ) at the higher c o n c e n t r a t i o n s . L i k e w i s e , most sour s o l u tions (organic and i n o r g a n i c acids) were r e j e c t e d by c h i c k e n s . The e f f e c t s of p r i o r experience on t a s t e preference behavior o f the fowl was demonstrated (25) by the f a c t t h a t ascending versus descending s e r i e s o f preference t e s t concentrations y i e l d v a s t l y d i f f e r e n t data. The importance o f p r i o r experience was confirmed by Davidson (83) who reported on preference behavior o f various b i r d species toward b e r r i e s and seeds i n natural s e t t i n g s . He has suggested that low consumption of a new food item by many b i r d species f o r 1-10 days i s o f no p a r t i c u l a r s i g n i f i c a n c e . Thus, c e r t a i n avian species may be extremely slow to change t h e i r normal feeding h a b i t s . D e t a i l e d and systematic l a b o r a t o r y f l a v o r preference e v a l u ations i n w i l d b i r d s are somewhat s c a n t . Working with f e r a l

In Flavor Chemistry of Animal Foods; Bullard, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

30

FLAVOR

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FOODS

pigeons, Duncan (84) demonstrated that both a c e t i c and hydroc h l o r i c acids (sour to man) produce pronounced r e j e c t i o n even at low concentrations ( e . g . , .005N a c e t i c a c i d ) . With sodium c h l o r i d e , Duncan has i n d i c a t e d that the preference - aversion f u n c t i o n i s s i m i l a r to that found in the r a t . That i s , preference was i n d i c a t e d at wt/vol concentrations below 1.0% and the b i r d s tended to r e j e c t concentrations greater than 1.0%. Some o f the d i s c r e p a n c i e s found f o r preference behavior o f the same general species ( e . g . , c h i c k e n s , q u a i l , pigeons) could be a f u n c t i o n of d i f f e r e n t methods o f measuring p r e f e r e n c e . Gent i l e (85), f o r example, found that chickens p r e f e r r e d c e r t a i n concentrations of sucrose and glucose (1-5%) when b r i e f - e x p o s u r e (< 3 minutes) preference t e s t s are used. G e n t i l e b e l i e v e s that the Kare and Medway (86) preference data f o r carbohydrates r e f l e c t not only t a s t e but a l s o p o s t i n g e s t i o n a l f a c t o r s . As summarized by Wenze u l i ( e . g . , quinine h y d r o c h l o r i d e e r a l l y r e j e c t e d by the pigeon, q u a i l , c h i c k e n , and Great T i t . Sour s t i m u l i ( a c e t i c and h y d r o c h l o r i c acids) are a l s o r e j e c t e d by most b i r d species t e s t e d except f o r q u a i l that p r e f e r s l i g h t l y sour tastes. The Great T i t and q u a i l a l s o p r e f e r glucose, but the pigeon and chicken are l e s s p r e d i c t a b l e . Using more natural t a s t e s t i m u l i , Yang and Kare (87) reported that c e r t a i n arthropod defensive s e c r e t i o n s could g r e a t l y a f f e c t red-winged b l a c k b i r d preference behavior. Both sal i c y ! a l d e h y d e and p-benzoquinone were r e j e c t e d at very low concentrations (0.025% wt/vol) in water s o l u t i o n s . The data may i n d i c a t e c e r t a i n i n s e c t species gain p r o t e c t i o n from b i r d p r é d a t i o n by v i r t u e of t h e i r chemical s e c r e t i o n s that t a s t e unpleasant or i r r i t a t i n g to b i r d s . Brower's (88) mimicry model o f s e l e c t i v e p r é d a t i o n proposes that b i r d s are i n i t i a l l y i n f l u e n c e d to feed on i n s e c t s by such f a c t o r s as movement, s i g h t , shape, and other nontaste cues. Howe v e r , a f t e r an unpleasant experience with these i n s e c t substances, c e r t a i n aspects o f the food item become avoided. These can i n c l u d e t a s t e as well as c o l o r , shape, t e x t u r e , and s i z e o f s p e c i f i c insects. Some i n t e r e s t i n g p o s s i b i l i t i e s f o r the development o f improved b i r d r e p e l l e n t chemicals are discussed along these l i n e s by Rogers (89). L i t t l e work has been done i n an attempt to i n c r e a s e b a i t acceptance by pest b i r d s . Bui l a r d (90) has provided some evidence that b a i t formulation could be a c r i t i c a l f a c t o r i n b i r d - b a i t i n g programs. Surface-coated grain b a i t s ( c o r n , wheat, or oats) cont a i n i n g a b i r d r e p e l l e n t (methiocarb) were shown to vary g r e a t l y in chemical concentration ( c . v . = 21-48%). Uniformly mixed and t a b l e t e d b a i t s had much l e s s chemical concentration v a r i a b i l i t y ( c . v . = 4.6-8.0%). These data would i n d i c a t e t h a t a more uniform r e p e l l e n t dosage can be achieved with t a b l e t e d m a t e r i a l . Most of the emphasis i n research and development o f b i r d cont r o l agents has been d i r e c t e d at the development of improved r e p e l l e n t s (89). Recent e f f o r t s i n t o t h i s research include an

In Flavor Chemistry of Animal Foods; Bullard, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

2.

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31

e v a l u a t i o n of the mode o f a c t i o n o f commercially marketed " b i r d r e s i s t a n t " v a r i e t i e s o f sorghum (91). Although the data are not y e t f u l l y analyzed, i t appears that a rough negative c o r r e l a t i o n e x i s t s between polyphenolic content of the seed v a r i e t y and seed preference by quelea and red-winged b l a c k b i r d s . It i s hoped that the research i n t o the natural secondary plant substances [ e . g . , a l k a l o i d s (92) and a s t r i n g e n t s (93)] w i l l e v e n t u a l l y allow improved crop p r o t e c t i o n from d e s t r u c t i v e b i r d s p e c i e s . It i s a l s o hoped that t h i s research can lead to the development o f improved b i r d r e s i s t a n t grain v a r i e t i e s or the i s o l a t i o n and eventual marketing of extremely s a f e , e f f e c t i v e , and low-cost t a s t e r e p e l l e n t chemicals. Empirical Screening of F l a v o r i n g Agents, Enhancers, and Spices O b v i o u s l y , the f l a v o expected to research a l (nutritional c a l , b e h a v i o r a l , e t c . ) that w i l l a f f e c t the preference behavior of a given b i r d o r mammal. Some researchers have t h e r e f o r e taken an e m p i r i c a l screening approach to the problem. For example, H i l k e r e t a l . (94) attempted to a l t e r food preferences o f rats using a mixture of black pepper, c l o v e s , and cinnamon each at 0.5% concentration. The s p i c e d d i e t was fed to weanling rats f o r 5 weeks followed by a 2-week f r e e - c h o i c e p e r i o d . The young rats consumed s i g n i f i c a n t l y l e s s of the s p i c e d d i e t compared with the untreated d i e t , whereas adults showed no p a r t i c u l a r preference between the d i e t s . Spices and f l a v o r i n g s are thought (95) to stimulate a p p e t i t e and g a s t r o i n t e s t i n a l readiness f o r r e c e i v i n g food. However, a more recent (23) viewpoint on t h i s s u b j e c t i s that the f l a v o r s o f a d d i t i v e s p r i m a r i l y serve as d i s t i n c t i v e t a s t e and odor cues f o r the s a f e t y and f a m i l i a r i t y with c e r t a i n foods eaten by man. I f t h i s viewpoint i s c o r r e c t , the screening of f l a voring agents and spices to increase b a i t consumption by rodents in a g r i c u l t u r a l areas may be v a l u e l e s s . B a i t a d d i t i v e s that enhance n a t u r a l l y p r e f e r r e d food f l a v o r s could o f f e r a p o t e n t i a l means o f improving b a i t i n g programs by a t t r a c t i n g l a r g e r numbers o f rats to the b a i t s and by i n c r e a s i n g t o x i c b a i t consumption. We have not been able to demonstrate that p r o t e i n f l a v o r enhancers ( i . e . , 5 ' - r i b o n u c l e o t i d e s ) (96, 9 7 ) , p o t e n t i a t o r s such as monosodium - L - glutamate, or sweetness enhancers added to ground r i c e b a i t have any p a r t i c u l a r l y strong e f f e c t s on the choice behavior o f r i c e f i e l d r a t s from the P h i l i p pines (Table II). These f i n d i n g s tend to support the views that the t a s t e and odor perceptual systems of man versus rodent are v a s t l y d i f f e r e n t (1, 9 8 ) , and t h a t man's acceptance o f some o f these products may~be, i n large p a r t , c u l t u r a l l y determined (23). Food odors as area a t t r a c t a n t s to rodents f o r c o n t r o l purposes have been postulated f o r many years (99^ 100, 101, 103). R e i f f (99) took a r a t h e r a n a l y t i c approach to the f l a v o r p r e f e r ence prôFlem i n rodents by i n v e s t i g a t i n g c e r t a i n chemical compounds

In Flavor Chemistry of Animal Foods; Bullard, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

32

FLAVOR

Table II.

No.

1 2 3 4 5 6 7 8 9 10 11 12

CHEMISTRY

OF ANIMAL

Mean percent preference ± S.E.M. f o r 12 b a i t each t e s t e d at three c o n c e n t r a t i o n s .

Addi t i v e name

Concentrât!on C-j C

a

V-50 Dried humeri Zymino Canned food flavor Cereal f l a v o r Vegamine #1 Vegamine #28 Vegamine #69 V 84T Soy sugar V e l t o l plus Sugar

Control

FOODS

additives

b

C

47.4±1.0 49.2±1.0 50.3±1.1

47.0±2.7 45.1+5.6 42.0±4.8

45.3±4.2 39.9±5.3 47.3±6.8

29.7±6.5 48.6±4.8 43.H3.8

51.7±1.8 50.0+0.8 48.7±2.0 50.7±1.2 52.1+0.8 48.2±0.8 50.1+1.0 51 .9±1.7 51.7±0.8

42.1±4.2 51.1+1.7 46.3+2.5 43.8±4.7 43.1±6.3 44.1±2.1 46.H5.1 51.8±0.7 57.6±3.2

30.6+7.8 39.4±5.0 50.1+2.9 41.5±6.3 41,4±4.9 38.0±4.0 52.9±2.1 46.1±0.8 51.9+2.6

38.6±4.8 37.7+5.2 38.3±6.6 49.6±5.2 44.6±6.1 42.H3.1 49.8±4.0 44.7±1.7 60.2±3.7

C

A d d i t i v e numbers 1-10 are p r o t e i n h y d r o l y s a t e s ; number 11 i s a sweetness enhancer; number 12 i s a n u t r i t i v e sweetener. A d d i t i v e numbers 1-8 were t e s t e d C = 1.2%; number 9 t e s t e d a t : C-, numbers 10 and 11 t e s t e d a t : Ci = number 12 a t : Cj = 10%, C = 2 0 % ,

b

3

2

c

at: C-j = 0.3%, C = 0.6%, = .15%, Co = 0.3%, Co = 0.75%; .10%, C = 0.2%, Co = 0.4%; C = 40%. 2

2

3

Ρ (C3 > Control) < .05.

In Flavor Chemistry of Animal Foods; Bullard, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

2.

SHUMAKE

Food Preference

Behavior

33

commonly found i n food items eaten by rats and mice. C e r t a i n t e r t i a r y amines, e s s e n t i a l o i l s , and aldehydes are found n a t u r a l l y in meats, vegetables, cereal g r a i n s , and d a i r y products known to be r e a d i l y eaten by r a t s . Although these elemental f l a v o r compounds can d i r e c t l y c o n t r i b u t e to the t o t a l f l a v o r o f c e r t a i n food items f o r r a t s , t h e i r combined a c t i o n i s g e n e r a l l y much stronger than the a c t i o n o f any s i n g l e compound. We have found t h i s same general e f f e c t with r i c e f i e l d rats (18). Of 20 candidate food odor compounds evaluated with an automated odor t e s t device (102), none produced as much i n v e s t i g a t i o n as the food odor (Purina Laboratory Chow) a s s o c i a t e d with t h e i r normal l a b o r a t o r y d i e t (see Table III). B u l l (103) came to a s i m i l a r c o n c l u s i o n i n h i s e v a l uation of more general food odors ( f i s h , b e e f , dog f o o d , coconut o i l ) and f l a v o r i n g agents ( r a s p b e r r y , aniseed o i l ) . Even with " r e p e l l e n t " odors, strong odor r e p e l l e n t e f f e c t s were only observed when the a c t i v food i n the hoppers. A g a i n along with texture and p a r t i c l e s i z e , proved to be more i n f l u e n t i a l on r a t feeding behavior than the presence or absence o f novel food odors. For w i l d rodents, and perhaps o t h e r macro-osmic mammals, food odor a t t r a c t a n t s are h e a v i l y dependent upon the animals' past experience i n t a s t i n g , i n g e s t i n g , and d e r i v i n g n u t r i t i o n a l b e n e f i t s from the food. Hansson (104) noted t h a t f i e l d v o l e s , bank v o l e s , and wood mice showed increased gnawing on s t i c k s impregnated with various vegetable o i l s . Part o f t h i s response appeared to be r e l a t e d to texture changes i n the wood as the o i l s penetrated i t . Some of the response, however, a l s o appeared to be r e l a t e d to the presence of c e r t a i n long-chain f a t t y acids ( e . g . , o l e i c , l i n o l e i c ) i n the oils. We have compared the e f f e c t s o f adding 10% vegetable or g r a i n o i l s to r i c e b a i t with independent groups o f R_. jr. mindanensis. Rats i n a l l groups showed r e l i a b l e preference f o r soybean, c o r n , peanut, l i n s e e d , palm k e r n e l , s a f f l o w e r , coconut, and r i c e o i l over untreated b a i t m a t e r i a l . As can be seen i n Table IV, Freon-11 e x t r a c t e d r i c e o i l produced the most c o n s i s t e n t e f f e c t on r i c e b a i t consumption. A l l o i l s changed the t e x t u r e as well as t a s t e and odor of the b a i t . This texture change e f f e c t was evaluated by comparing the preference responses f o r the s a f f l o w e r o i l ( h i g h - o l e i c type) with the other o i l s s i n c e t h i s material has been reported (105) to be odorless and t a s t e l e s s to man. Supposedly, even with a very low t a s t e or odor component to r a t s , s a f f l o w e r o i l s t i l l produced almost 90% preference a f t e r 6 exposure t e s t days. The r i c e o i l was the most c o n s i s t e n t l y p r e f e r r e d mater i a l , probably because i t acted to i n t e n s i f y the f l a v o r of a n o r mally h i g h l y p r e f e r r e d and f a m i l i a r food ( i . e . , r i c e ) . Intensifying

F l a v o r with Extracted or V o l a t i l i z e d Compounds

The e m p i r i c a l screening approach c a n , at times, lead to s u c c e s s f u l development of improved animal food f l a v o r i n g agents as

In Flavor Chemistry of Animal Foods; Bullard, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

34

FLAVOR

Table III.

CHEMISTRY

OF ANIMAL

FOODS

Odor preference test results when 20 candidate mate­ r i a l s were compared with investigation time toward the familiar food odor (Purina Laboratory Rat Chow). Percent response

Candidate food odor attractan Soybean o i l Isovaleric aldehyde N^butyldiethanolamine N-proplyamine Peanut o i l 2-Furaldehyde Linseed o i l Εthy 1 diethanolami ne Sassafrass o i l Dihydroxyethy1 ani1ine Wintergreen o i l Corn o i l Hexanoic acid N^octylamine N-amylamine Isobutylamine Cod l i v e r o i l ( n-p ropy 1 ) -b enzy 1 ami ne Valerone JN-hexylamine

94.6 94.3 87.5 80.0 80.0 77.5 76.2 75.6 74.4 72.5 72.2 67.5 65.9 61.9 60.0 52.4 50.0 40.0 36.4 35.6

+ + ± + + + + + + + + + + + + + + + + +

In Flavor Chemistry of Animal Foods; Bullard, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

23.0 21.6 38.6 56.4 18.3 31.3 24.1 11.5 23.9 58.8 16.9 16.3 25.9 26.6 42.9 19.7 19.3 28.1 4.5 3.6

In Flavor Chemistry of Animal Foods; Bullard, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

Soybean

54.6±32.8 60.8±29.3 76.6+36.1 75.0±34.2 72.6±35.1 79.3+36.0

69.8±33.9

1 2 3 4 5 6

Mean +S.D.

73.0±36.7

61.8±30.3 67.1+34.8 76.8±38.6 75.3+38.6 78.8±38.9 78.2±39.1

Corn

80.U15.6

55.6±24.9 79.9±12.3 83.0+12.6 84.9±15.0 88.H13.9 89.3±14.8

Peanut

85.4±17.3

71.3+21.6 88.1±14.4 88.4±12.9 90.3±17.2 88.5±18.0 86.0±19.5

Linseed

Oil

74.0+14.9

64.8±19.3 78.8+12.4 77.5+11.7 79.7±10.3 71.0+15.2 72.3+20.2

Palm kernel

81.4±26.0

65.9±38.4 80.2±23.7 82.3±25.0 82.3±25.0 88.3+21.5 89.6+22.2

Higholeicsafflower

64.2±31.2

25.9±32.8 66.8±35.8 71.U38.5 69.9±35.7 87.2± 7.3 64.H37.1

Coconut

94.0± 8.0

83.9± 6.8 97.9+ 2.3 98.3± 3.9 96.2+ 7.0 93.8±14.9 94.U13.1

Freon-11 extractedrice o i l

Mean ± S.D. percent preference o f r i c e f i e l d r a t s f o r r i c e c o n t a i n i n g o i l - t r e a t e d versus untreated r i c e .

Day

Table IV.

36

FLAVOR

CHEMISTRY

OF ANIMAL

FOODS

i n d i c a t e d above. However, a more d i r e c t approach to the problem involves the study of what the animal normally or n a t u r a l l y p r e fers i n i t s native h a b i t a t . This most p r e f e r r e d (or l e a s t p r e f e r r e d ) food i s then chemically analyzed i n conjunction with a s e r i e s of behavioral b i o a s s a y s . To i l l u s t r a t e t h i s approach, I have o u t l i n e d the f o l l o w i n g examples. Rice and Church (106) presented evidence that b l a c k - t a i l e d deer p r e f e r d i s t i l l e d water t r e a t e d with low concentrations o f water or ethanol e x t r a c t s from foods normally accepted by deer ( b i t t e r brush, D o u g l a s - f i r , and hemlock). In response to c e r t a i n organic acids commonly found i n plants browsed by ungulates, there were pronounced sex d i f f e r e n c e s . Bucks s t r o n g l y p r e f e r r e d the intermediate concentrations o f malic a c i d but showed weaker p r e f erence f o r s u c c i n i c and c i t r i c a c i d s . Does, on the other hand, showed weak to strong r e j e c t i o n of these same a c i d c o n c e n t r a t i o n s . This report i n d i c a t e d tha natural food sources o consummatory d r i n k i n g behavior. Mugford (107) reported that both the duration and the s i z e of meals eaten by domestic cats can be increased by p e r f u s i n g normally p r e f e r r e d meat odors (cooked r a b b i t ) through t h e i r maintenance d i e t (dry food). Le Magnen (108) and Larue (109) have a l s o noted that food odors can i n f l u e n c e feeding patterns in r a t s . For c e r t a i n predatory mammals such as foxes (65), food odors may e l i c i t food seeking regardless o f experience. For many other mammalian species [ e . g . , Norway rats ( 4 ) , polecats (66), and r o o f rats (58)] food f l a v o r preferences are~highly dependent upon experience with the food or prey s p e c i e s . As another example o f an a l t e r n a t e approach to e m p i r i c a l s c r e e n i n g , B u l l a r d and Shumake (110) evaluated e i g h t p o t e n t i a l f l a v o r components of whole g r a i n , uncooked r i c e with r i c e f i e l d rats in an attempt t o increase the p a l a t a b i l i t y o f ground r i c e baits. Rice bran o i l , the a c e t o n i t r i l e e x t r a c t o f r i c e bran o i l , the ether e x t r a c t of ground r i c e , endosperm, r i c e p o l i s h , r i c e bran, r i c e bran v o l a t i l e s , and whole grain r i c e v o l a t i l e s were evaluated as a d d i t i v e s to ground r i c e with a group o f 12 r a t s . A b r i e f exposure t a s t e preference device (18^, 111) was used i n these evaluations to ensure that p o s t i n g e s t i o n a l f a c t o r s r e l a t e d to c a l o r i c content or d i g e s t a b i l i t y would not confound preference test results. The whole grain r i c e v o l a t i l e s material was the only one found to r e l i a b l y i n c r e a s e both consumption and feeding time. In a l a t e r s e m i - f i e l d pen t e s t , r i c e v o l a t i l e s t r e a t e d b a i t was p r e f e r r e d (P < .05) over w h o l e - g r a i n , granulated, and soy bean o i l (1%) t r e a t e d r i c e . The e f f e c t of the r i c e v o l a t i l e s a d d i t i v e on t o x i c b a i t consumption was then evaluated (110). Two groups o f rats were given a choice between e i t h e r 0.2% z i n c phophide t r e a t e d r i c e and untreated r i c e , or 0.2% z i n c phosphide t r e a t e d r i c e with the r i c e v o l a t i l e s added and untreated r i c e . This l a t t e r group showed almost double the t o x i c r i c e b a i t consumption and a m o r t a l i t y o f

In Flavor Chemistry of Animal Foods; Bullard, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

2.

SHUMAKE

Food Preference

Behavior

37

88%. The c o n t r o l group r a t s (without r i c e v o l a t i l e s on t o x i c r i c e b a i t ) showed only 50% m o r t a l i t y (P < .005). This experiment i n d i c a t e d to us that f l a v o r s from a p r e f e r r e d food f o r r a t s could be f u r t h e r i n t e n s i f i e d to increase p a l a t a b i l i t y . T h i s increased p a l a t a b i l i t y had the advantage of i n c r e a s i n g t o x i c b a i t consumpt i o n , presumably because we were working with h i g h l y f a m i l i a r r i c e f l a v o r s p r e f e r r e d by r i c e f i e l d rats (112, 113). Conclusions I t appears to be u n l i k e l y t h a t food f l a v o r i n g agents, s p i c e s , and enhancers developed f o r humans w i l l e f f e c t strong and l o n g l a s t i n g preference behavior m o d i f i c a t i o n s i n b i r d s and mammals. Food f l a v o r f a m i l i a r i t y may be the s i n g l e , most important f a c t o r c o n t r o l l i n g food preference behavior o f w i l d species (8 £ 3 77) Mammals show highest acceptanc tend to sample small amount environment ( 4 ) . Some b i r d species are extremely r e s i s t a n t to changing t h e i r preference behavior f o r c e r t a i n food items (83), but there i s no compelling evidence proving t h a t food f l a v o r i s less important to b i r d s than to mammals (62, 85, 89). For mammals (and p o s s i b l y b i r d s a l s o ) , food t e x t u r e , p a r t i c l e s i z e , and feeder l o c a t i o n can sometimes produce as much, or more, e f f e c t than odor and t a s t e of food (103, 104). Preference t e s t r e s u l t s f o r a given f l a v o r a d d i t i v e and species w i l l vary with methodological f a c t o r s such as: length of t e s t , previous experience of the t e s t animal, and t e s t f l u i d or food base used. In g e n e r a l , t a s t e preference evaluations with standard compounds ( e . g . , NaCl, s u c r o s e , HC1, and quinine) do not y i e l d data that allow p r e d i c t i o n o f the kinds o f food f l a v o r s w i l d animals w i l l most r e a d i l y accept and p r e f e r i n a given ecosystem. Food habit s t u d i e s that take i n t o account the r e l a t i v e abundance of the food items under study, can sometimes y i e l d the most p r o ductive and p r e d i c t i v e i n f o r m a t i o n . Empirical screening as a method of searching f o r animal food a t t r a c t a n t s or r e p e l l e n t s can be an expensive e f f o r t and i s , at times, not worthy o f the r e s e a r c h e r ' s time. Attempts to c o r r e l a t e chemical s t r u c t u r e with r e p e l l e n t e f f e c t s e l i c i t e d by compounds from an e m p i r i c a l s c r e e n ing program i n rodents have l e d to some degree o f p r e d i c t i o n (114), but the e f f o r t and expense o f such a c t i v i t y may not be e a s i l y justified. C o r r e l a t i o n o f chemical s t r u c t u r e with b i o l o g i c a l l y a c t i v e compounds i s o l a t e d from natural sources ( e . g . , secondary plant substances) may be a more f r u i t f u l approach. Our approach to developing an improved and i n t e n s i f i e d r i c e f l a v o r f o r b a i t i n g rats with r i c e has been as f o l l o w s . F i r s t , the natural or normal food habits of the r i c e f i e l d r a t were evaluated under f i e l d c o n d i t i o n s (113). Second, t h i s preference response was then confirmed f o r r i c e under b r i e f - e x p o s u r e , two-choice l a b o r a t o r y t e s t c o n d i t i o n s . T h i r d , the most f a m i l i a r and p r e f e r r e d food, r i c e , was separated i n t o gross chemical components ( e . g . ,

In Flavor Chemistry of Animal Foods; Bullard, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

FLAVOR

38

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bran, endosperm, o i l , f l a v o r v o l a t i l e s , e t c . ) (110), and each o f these was s e p a r a t e l y added back to ground r i c e . A d d i t i o n a l behav­ i o r a l t e s t s were then conducted to evaluate p o t e n t i a l a p p l i c a t i o n of the most p r e f e r r e d f l a v o r component ( e . g . , i n c r e a s i n g t o x i c b a i t consumption, i n c r e a s i n g b a i t p a l a t a b i l i t y , and a t t r a c t i n g rats from a d i s t a n c e ) . I f a l l l a b o r a t o r y and s e m i - f i e l d e v a l u a ­ tions continue to be p o s i t i v e and c o n s i s t e n t , we w i l l then proceed to s m a l l - and l a r g e - s c a l e f i e l d t e s t i n g . A f t e r some p o s i t i v e con­ f i r m a t i o n from the f i e l d t e s t s , we w i l l then e i t h e r synthesize the natural r i c e v o l a t i l e s f l a v o r o r devise a commercial e x t r a c t i o n process f o r producing t h i s b a i t a d d i t i v e . The approach to developing improved b i r d r e p e l l e n t s can p r o ­ ceed along these same l i n e s . There are numerous seeds, b e r r i e s , and plants that b i r d s normally a v o i d . Part o f t h i s avoidance could be due to conditioned aversion (89) or to a v e r s i v e t a s t e (93). Regardless o f th involved i n the r e p e l l e n e x p l o i t a t i o n o f natural food items as a source f o r developing more a t t r a c t i v e or r e p e l l e n t animal food f l a v o r s w i l l c o n t i n u e . Acknowledgments Thanks are due to Mr. R. W. Bui l a r d and Dr. R. T . Sterner f o r reviewing e a r l y d r a f t s o f the manuscript. The author i s a l s o g r a t e f u l f o r the t e c h n i c a l a s s i s t a n c e o f Mr. K. A. Crane and Mr. S. E. Gaddis o f the Denver W i l d l i f e Research Center as well as personnel at the Rodent Research Center, Los Banos, The P h i l i p p i n e s . This work was supported, i n p a r t , with funds provided t o the U.S. Fish and W i l d l i f e S e r v i c e by the U.S. Agency f o r I n t e r n a t i o n a l Development under the p r o j e c t "Control o f Vertebrate Pests: Rats, B a t s , and Noxious B i r d s , " PASA RA(ID) 1-67.

Literature Cited 1. Kare, M. R. J. Agric. Food Chem. (1969) 17:677-680. 2. Watson, A. "Animal Populations in Relation to Their Food Resources," Blackwell Scientific Press, Oxford, 1970. 3. Le Magnen, J. "Handbook of Sensory Physiology," IV, Beidler, L. M., Ed., pp. 463-482, Springer-Verlag, Berlin, 1971. 4. Barnett, S. A. Brit. J. Anim. Behav. (1953) 1:159. 5. Crook, J. H. and Ward, P. "The Problems of Birds as Pests," Murton, R. K. and Wright, Ε. Ν., Eds., pp. 211-230, Academic Press, New York, 1968. 6. Ward, P. Ibis (1965) 107:173-214. 7. Geist, V. "Mountain Sheep," Univ. Chicago Press, Chicago, 1971. 8. Barnett, S. A. "The Rat: A Study in Behavior," Univ. Chicago Press, Chicago, 1975. 9. Kare, M. R. "The Physiological and Behavioral Aspects of Taste," Kare, M. R. and Halpern, B. P., Eds., pp. 6-15,

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36. Harris, L. J., Clary, J., Hargreaves, F. J. and Ward, A. Proc. Roy. Soc, B, Biol. Sci. (1933) 113:161-190. 37. Bernard, R. Α., Hal pern, B. P. and Kare, M. R. Proc. Soc. Exptl. Biol. Med. (1961) 108:784-786. 38. Bernard, R. A. and Halpern, B. P. J. Gen. Physiol. (1968) 52:444-464. 39. Hughes, B.O.and Wood-Gush, D. G. Physiol. Behav. (1971) 6:331-339. 40. Richter, C. P. Am. J. Physiol. (1936) 115:155-161. 41. Scott, E. M., Verney, E. L. and Morissey, P. D. J. Nutr. (1950) 41:173-186. 42. Wolf, G. and Steinbaum, E. A. J. Comp. Physiol. Psychol. (1965) 59:335-339. 43. Schmidt-Nielsen, K. Circulation (1960) 21:955-967. 44. Adair, E. R., Miller, Ν. E. and Booth, D. A. Commun. Behav. Biol. (1968) 2:25-37 45. Valenstein, E. S. (1967) 157:552-554. 46. Mailer, O. Proc. Soc. Exptl. Biol. Med. (1965) 119:199-203. 47. Piquard, F., Schaffer, A. and Haberey, P. Physiol. Behav. (1975) 15:41-46. 48. Booth, D. A. and Campbell, C. S. Physiol. Behav. (1975) 15:523-535. 49. Ter Haar, M. B. Horm. Behav. (1972) 3:213-219. 50. Pietras, R. J. and Moulton, D. G. Physiol. Behav. (1974) 12:475-491. 51. Wade, G. N. and Zucker, I. Physiol. Behav. (1970) 5:269273. 52. Valenstein, E. S., Kakolewski, J. W. and Cox, V. C. Science (1967) 156:942-943. 53. Hoff, L. A. Dissert. Abst. (1968) 28:4314. 54. Booth, D. A. and Davis, J. D. Physiol. Behav. (1973) 11: 23-29. 55. Mitchell, D., Beatty, Ε. T. and Cox, P. K. Behav. Biol. (1977) 19:206-216. 56. Young, P. T. and Kappauf, W. E. Amer. J. Psychol. (1962) 75:482-484. 57. SFumake, S. Α., Thompson, R. D. and Caudill, C. J. J. Comp. Physiol. Psychol. (1971) 77:489-494. 58. Jackson, W. B. Pest Cont.(1965)33:12,13,50,52. 59. Wagner, M. W. Psychol. Repts.(1971)29:799-804. 60. Ader, R. Bull. Psychon. Soc. (1973) 17253-254. 61. Pelz, W. E., Whitney, G. and Smith, J. C. Physiol. Behav. (1973) 10:263-265. 62. Wenzel, Β. M. "Avian Biology: III," Farner, D. S., King, J. R. and Parkes, K.C.,Eds., pp. 389-415, Academic Press, New York, 1973. 63. Kare, M. R. and Maller, O. J. Nutr. (1967) 92:191-196. 64. Pearson,O.P. J. Anim. Ecol. (1966) 35:217-233. 65. Macdonald, D. W. Mammal Rev. (1977) 7:7-23.

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Apfelback, R. Zeit. Tierpsychol. (1973) 33:270-273-. Krishnakumari, M. K. Pest Control (1973) 41:36, 38, 43. Bronson, G. J. Comp. Physiol. Psychol. (19SS") 62:162-164. Capretta, P. J. and Rawls, L. H. J. Comp. Physiol. Psychol. (1974) 86:670-673. Bronstein, P. M. and Crockett, D. P. Behav. Biol. (1976) 18:387-392. Cornwell, C. A. Behav. Biol. (1976) 17:131-137. Galef, B. G., Jr., and Sherry, D. F. J. Comp. Physiol. Psychol. (1973) 83:374-378. Galef, B. G., Jr., and Henderson, P. W. J. Comp. Physiol. Psychol. (1972) 78:213-219. Galef, B. G., Jr., and Clark, M. M. J. Comp. Physiol. Psychol. (1972) 78:220-225. Leon, M. and Moltz H Physiol Behav (1971) 7:265-277 Leon, M. and Moltz Leon, M., Galef, B Physiol Behav. (1977) 18:387-391. Galef, B. G., Jr., and Heiber, L. J. Comp. Physiol. Psychol. (1976) 90:727-739. Kuehnert, G. Plant Prot. News (Manila) (1976) 5:24-27. Reichman,O.J. J. Mammal. (1975) 56:731-751. Turkowski, F. J. J. Wildl. Manage. (1975) 39:748-756. Fellows, D. P. and Sugihara, R. T. Hawaiian Plant Rec. (1977) 59:67-86. Davidson, V. E. Audubon (1962) 64:346-350. Duncan, C. J. Anim. Behav. (1960) 8:54-60. Gentile, M. J. "Neural and Endocrine Aspects of Behavior in Birds," Wright, P., Caryl, P. G. and Vowles, D. M., Eds., pp. 305-318, Elsevier Scientific Publishing Co., Amsterdam, 1975. Kare, M. R. and Medway, W. Poult. Sci. (1959) 38:1119-1127. Yang, R. S. H. and Kare, M. R. Ann. Entomol. Soc. Am. (1968) 61:781-782. Brower, L. P. Sci. Am. (1969) 220:22-29. Rogers, J. G., Jr. "Flavor Chemistry of Animal Foods," Bullard, R. W., Ed., American Chemical Society, Washington (in press). Bullard, R. W. J. Wildl. Manage. (1970) 34:925-929. Bullard, R. W. Personal communication, Denver Wildlife Research Center, 1977. McKey, D. Am. Nat. (1974) 108:305-320. Bate-Smith, E. C. "Phytochemistry and Ecology: Proceedings of the Phytochemical Society Symposium," Harborne, J. B., Ed., pp. 45-56, Academic Press, London, 1972. Hilker, D. M., Hee, J., Higashi, J., Ikehara, S. and Paulsen, E. J. Nutr. (1967) 91:129-131. Mukerji, B. Fed. Proc. (1961) 20:247. Yamaguchi, S., Yoshikawa, T., Ikeda, S. and Ninomiya, T. Agri. Biol. Chem. (1968) 32:797-802. Sato, M. and Yamashita, S. Jap. J. Physiol. (1965) 15:

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570-578. 98. Halpern, B. P., Bernard, R. A. and Kare, M. R. J. Gen. Physiol. (1962) 45:681-701. 99. Reiff, M. Acta Tropica (1956) 13:289-318. 100. Steinbrecher, W. Zeit. Ange. ZooT7 (1962) 49:301-349. 101. Long, C. J. and Tapp, J. T. Psychon. Sci. (1967) 1:17-18. 102. Shumake, S. Α., Thompson, R. D. and Bullard, R. W. Behav. Res. Methods Instrum. (1973) 5:279-282. 103. Bull, J. O. Proc. Fifth Vert. Pest Conf. (1972) 5:154-160. 104. Hansson, L. OIK0S (Copenhagen) (1973) 24:417-421. 105. Flath, R. Α., Forrey, R. R. and Guadagni, D. G. J. Agric. Food Chem. (1973) 21:948-952. 106. Rice, P. R. and Church, D. C. J. Wildl. Manage. (1974) 38:830-836. 107. Mugford, R. A. "Chemical Senses and Nutrition," Kare, M. R. and Mailer,O., (In press). 108. Le Magnen, J. Probl. Actuels d'Endocrinol. Nutr. (1963) 7:147-171. 109. Larue, C. G. and Le Magnen, J. Physiol. Behav. (1972) 9:817-821. 110. Bullard, R. W. and Shumake, S. A. J. Wildl. Manage. (1977) 41:290-297. 111. Thompson, R. D. and Grant, C. V. J. Exp. Anal. Behav. (1971) 15:215-220. 112. Shumake, S.A. "Chemical Signals in Vertebrates," MüllerSchwarze, D. and Mozell, M. M., Eds., pp. 357-376, Plenum Publishing Co., New York, 1977. 113. Tigner, J. R. Ph.D. Thesis (1972) University of Colorado. 114. Bowles, W. Α., Adomaitis, V. Α., De Witt, J. B. and Pratt, J. J., Jr. "Relationships between Chemical Structure and Rat Repellency. II. Compounds Screened between 1950 and 1960," U.S. Army Natick Lab. Tech. Rep. (75-11-FEL), Natick, Mass., 1974. RECEIVED October 25, 1977.

In Flavor Chemistry of Animal Foods; Bullard, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

3 Methodology of Behavioral Testing Associated with Development in Animal Foods JAMES C. SMITH and MICHAEL E. RASHOTTE Department of Psychology, The Florida State University, Talahassee, FL 32306 New animal foods shoul competitive, and readily accepte y target-anima population These goals are typically approached by drawing upon the expertise of nutritionists, economists and flavor chemists. It is our contention that animal food development can also benefit from the expertise of those psychobiologists whose interests relate to variables which influence food acceptance by animals. The present paper will discuss the nature of the psychobiologists approach to the problem of food acceptance and will outline some findings which we feel illustrate the potential contribution of this group of scientists to the development of animal foods. At the 2nd International Symposium on Olfaction and Taste, Dr. Carl Pfaffmann and his collaborators presented a good example of the psychobiological perspective on the study of food preferences in animals (l). They pointed out that the key questions concern the variables which lead an animal to initiate, maintain and terminate behavior directed towards nutritive and non-nutritive substances. They classified these variables as physiological and behavioral. The physiological variables included : (l) afferent stimulation from olfactory gustatory and somatosensory receptors in the head, (2) both immediate and long-term consequences of ingestion (i.e., sensory and/or metabolic effects), and (3) the interaction of afferent stimulation and postingestive consequences which modify preference and ingestive behavior. The behavioral variables are concerned primarily with the arrangement of the preference test situation itself, which under many circumstances, may profoundly affect reactions to the food stimuli being tested. Our principal emphasis here will be on the behavioral variables although we will devote some attention to interactions of the physiological and behavioral variables. It will be evident that the experimental evidence on which our discussion is based is wholly derived from studies with common laboratory species (e.g., rats, pigeons) and from the study of only a few foods. '

© 0-8412-0404-7/78/47-067-043$10.00/0 In Flavor Chemistry of Animal Foods; Bullard, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

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Despite the present absence of data from non-laboratory species and the l i m i t e d number of foods s t u d i e d , we are confident t h a t the methodological p o i n t s we make are b r o a d l y a p p l i c a b l e i n the t e s t i n g of food acceptance. The most obvious way t o assess a c c e p t a b i l i t y i s t o employ an i n g e s t i o n a l method i n which a food i s presented t o a group of animals and they are allowed t o consume i t f o r a p e r i o d of time. The i n g e s t i o n a l method i s f a m i l i a r as a technique f o r a s s e s s i n g the a c c e p t a b i l i t y of animal foods and, i n f a c t , the u l t i m a t e a r b i t e r of a c c e p t a b i l i t y i s the manner i n which a food i n i t i a t e s , maintains and terminates i n g e s t i o n a l behavior. N e v e r t h e l e s s , a second method f o r the study of food acceptance has evolved from the work of p s y c h o b i o l o g i s t s concerned w i t h l e a r n i n g processes i n animals, the i n s t r u m e n t a l method. In t h i s method the i n d i c a t o r of a c c e p t a b i l i t y i s a motor response which the animal i s r e q u i r e d to perform before i t gain l i z a t i o n of e i t h e r of thes c e r t a i n v a r i a b l e s be recognized. In the present paper we w i l l review the most important of these v a r i a b l e s f o r each methodology. I n g e s t i o n a l Method I t probably should be p o i n t e d out t h a t animal psychophysics d i f f e r s from human psychophysics i n one profound way. I f we want to know whether a human detects a food substance, d i s c r i m i n a t e s one food substance from another or p r e f e r s one food substance over another, we ask the person. We have v e r b a l r e p o r t as an i n d i c a t o r response. With animals we have no v e r b a l r e p o r t , so i n f e r d e t e c t i o n , d i s c r i m i n a t i o n , a c c e p t a b i l i t y and preference from the non-verbal behavior of the animal. The most common measure r e s u l t i n g from these non-verbal behaviors i s the amount of food i n g e s t e d . The i n g e s t i o n of foods by animals depends on a v a r i e t y of p h y s i o l o g i c a l and b e h a v i o r a l v a r i a b l e s . P.T. Young (2) has s t a t e d t h a t "food acceptance i s a complex process r e g u l a t e d by at l e a s t f o u r groups of determinants...organic c o n d i t i o n s , p e r i p h e r a l s t i m u l a t i o n , previous experience and b o d i l y c o n s t i t u t i o n " . Pfaffmann (l_) has p o i n t e d out the importance of both the immediate and long term "organic c o n d i t i o n s " and the i n t e r a c t i o n of these s t a t e s w i t h the p e r i p h e r a l s t i m u l a t i o n . He has a l s o s t r e s s e d b e h a v i o r a l v a r i a b l e s i n c l u d i n g the past h i s t o r y of the animal and the importance of the arrangement of the preference t e s t s i t u a t i o n i t s e l f . B u i l d i n g on the statements of Pfaffmann (l_) and Young (_2) , we have o u t l i n e d i n Table I what seems t o us to be the important determinants of the i n g e s t i o n of foods. We have s e l e c t e d only a l i m i t e d amount of data from t h i s and other l a b o r a t o r i e s t o i l l u s t r a t e the manner i n which these f a c t o r s may i n f l u e n c e i n g e s t i o n of foods. w e

B o d i l y C o n s t i t u t i o n . L i t t l e need be s a i d here. I t i s w i d e l y recognized t h a t t h e r e are profound d i f f e r e n c e s among species i n

In Flavor Chemistry of Animal Foods; Bullard, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

SMITH

Methodology

AND RASHOTTE

Table I .

of Behavioral

Factors i n f l u e n c i n g the a c c e p t a b i l i t y o f food.

I.

Bodily Consitiution I n t e r and i n t r a species d i f f e r e n c e s

II.

Organic c o n d i t i o n s Deprivation states S p e c i a l organic s t a t e s Immediate consequences o f i n g e s t i o n Long term consequences o f i n g e s t i o n

III.

Testing

Peripheral Stimulation Taste Olfaction Touch Vision Audition

IV.

V.

VI.

I n t e r a c t i o n o f P e r i p h e r a l S t i m u l a t i o n and Organic Conditions

Previous Experience Learned Preferences

and

Aversions

Conditions o f the a c c e p t a b i l i t y t e s t S i n g l e Food

M i l t i p l e Food

Long term t e s t

Short term t e s t

In Flavor Chemistry of Animal Foods; Bullard, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

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food i n g e s t i o n , d i e t a r y needs and t a s t e s . The "sweet t o o t h " of the r a t i s not t o "be found i n the c a t . In a d d i t i o n , one need not look f a r t o f i n d vast d i f f e r e n c e s between s t r a i n s of the same species. The C5T and the DBA mice provide good examples of d i f ferences i n i n t a k e s of s a c c h a r i n , a l c o h o l and morphine (3.)· Organic C o n d i t i o n s . One of the most widely s t u d i e d i n f l u ences on food i n g e s t i o n i s the d e p r i v a t i o n s t a t e of the animal. Inferences we would make about preferences are s t r o n g l y i n f l u enced by the l e n g t h of time the animal has gone without food and water. For example, under a v a r i e t y of c o n d i t i o n s sucrose preferences i n c r e a s e as a f u n c t i o n of d e p r i v a t i o n (h). In our own l a b o r a t o r y we have r e c e n t l y completed a study of food preferences i n the pigeon. A non-deprived pigeon shows approximately equal i n t a k e of Canadian peas and white m i l l e t i n a 2k hour 2 pan t e s t . B i r d s deprived t o Q0% o dian peas i n a short ter S p e c i a l Organic S t a t e s . Pregnancy, l a c t a t i o n , experimental s t a t e s induced by s u r g i c a l o p e r a t i o n s , implanted e l e c t r o d e s , drugs e t c . , a l l i n f l u e n c e acceptances of foods and l i q u i d s . Immediate Consequences of I n g e s t i o n . There are a number of consequences of i n g e s t i n g food t h a t are immediate and do not f a l l i n the category of the longer term metabolic s t a t e s of the animal. In 1952, McLeary (jO d i f f e r e n t i a t e d between t a s t e and postingest i o n f a c t o r s i n f l u e n c i n g the i n g e s t i o n of sucrose and concluded t h a t i n f i v e minutes or l e s s , the animal has already committed i t s e l f t o t a k i n g some p a r t i c u l a r volume of the s o l u t i o n . His evidence l e d him t o conclude t h a t hypotonic f l u i d i s r a p i d l y withdrawn i n t o the stomach f o l l o w i n g i n g e s t i o n of h i g h concent r a t i o n s of sugar and t h i s t r a n s i t o r y hypotonic s t a t e i s the p h y s i o l o g i c a l mechanism which leads t o l i m i t i n g the f u r t h e r i n g e s t i o n of sugar. More r e c e n t l y , Puerto et a l . , {6), have shown t h a t when a n u t r i e n t i s intubated i n t o the r a t ' s stomach, as the r a t i n g e s t s a n o n - n u t r i t i v e s o l u t i o n , the animal w i l l develop a preference f o r a f l u i d p a i r e d w i t h t h a t i n t u b a t i o n w i t h i n a 10 minute s e s s i o n . The mechanism u n d e r l y i n g t h i s e f f e c t i s unknown. V a l e n s t e i n (j) has shown t h a t d r i n k i n g 0.25$ sodium s a c c h a r i n p o t e n t i a t e d the e f f e c t of i n s u l i n i n r a t s . The r e s u l t s of t h i s experiment show t h a t although s a c c h a r i n i s not n u t r i t i v e i t i s not p h y s i o l o g i c a l l y i n e r t . I t seems l i k e l y the t a s t e of the sweet s a c c h a r i n i n i t i a t e s a " p r e p a r a t i v e metabolic r e f l e x " i . e . , p o s s i b l y i n s u l i n r e l e a s e d i n response t o the sweet t a s t e of the s a c c h a r i n . F i n a l l y , Kare (8_) at the T h i r d I n t e r n a t i o n a l Symposium on O l f a c t i o n and Taste presented data which suggests a d i r e c t pathway from the o r a l c a v i t y t o the b r a i n . Glucose l a b e l e d w i t h l^C a p p l i e d t o the o r a l c a v i t y f o r as short as h minutes went by a n o n - c i r c u l a t o r y route t o the b r a i n i n about h a l f of the animals t e s t e d . This r a p i d response could be r e l a t e d t o food-intake

In Flavor Chemistry of Animal Foods; Bullard, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

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Testing

metering. Long Term Consequences o f I n g e s t i o n . As we s h a l l see soon, there i s a profound d i f f e r e n c e i n the i n f e r e n c e we would make about the a c c e p t a b i l i t y o f sugars i f we use a short term ( l e s s than one hour) o r a long term ( u s u a l l y about one day) t e s t . This d i f f e r e n c e r e s u l t s from long term consequences of i n g e s t i o n which can operate i n the long term t e s t . C o l l i e r and B o l l e s (g.) s t a t e t h a t "constant c a l o r i c i n t a k e and balance o f d i e t a r y components, suggest t h a t the t y p i c a l preference t e s t paradigm should be r e ­ examined i n terms o f the r o l e o f the t e s t item i n the t o t a l nu­ t r i t i o n a l economy o f the animal". P e r i p h e r a l S t i m u l a t i o n . This i s Young's term {2) and he i s r e f e r r i n g t o the r o l e o f "head r e c e p t o r s " ( t a s t e , s m e l l , t o u c h , v i s i o n , a u d i t i o n ) i n meterin be the more important o nore the others. The odor o f a food and i t s t e x t u r e c e r t a i n l y w i l l be i n v o l v e d i n a t l e a s t the i n i t i a t i o n o f e a t i n g . I n some species v i s i o n may a l s o p l a y an important r o l e . I n the e x p e r i ­ ment r e f e r r e d t o e a r l i e r where we s t u d i e d pigeons' food preference and found t h a t the deprived b i r d always chose Canadian peas over m i l l e t , we were able t o a b o l i s h t h a t preference by crushing the Canadian peas so they were the same s i z e as the m i l l e t . I t seems most c e r t a i n t h a t v i s i o n plays the c r i t i c a l r o l e i n the pigeon's choice o f g r a i n s . So much emphasis has been placed on the "body wisdom" o f the l a b o r a t o r y - a n i m a l and how i t s e l e c t s an a p p r o p r i a t e l y balanced d i e t , one sees l i t t l e evidence i n the l i t e r a t u r e t h a t an animal might "go out t o dinner and eat f o r fun". C o l l i e r and B o l l e s ' ( .1 M. Because these are long term t e s t s , long term p o s t i n g e s t i o n a l f a c t o r s are o p e r a t i n g . C o l l i e r (g_) would say t h a t i f the r a t i s d r i n k i n g f o r s o l u t e or c a l o r i e s i t i s more e f f i c i e n t t o d r i n k from the higher concentrations suggesting a " p r i n c i p l e of l e a s t consummatory e f f o r t . " With the sugars, the s o l u t i o n of choice appears t o be d i f ­ f e r e n t w i t h the one b o t t l e and the two b o t t l e t e s t s . With sac­ c h a r i n , a non n u t r i t i v e substance, t h i s d i f f e r e n c e between the one and two b o t t l e t e s t s i s not apparent. T y p i c a l s a c c h a r i n i n t a k e s i n a one b o t t l e t e s t (e.g., 23) are presented i n F i g u r e 2. In two b o t t l e t e s t s we p a i r e d s a c c h a r i n concentrations of .03%, 0.1%, 0.3%, 0.9%, and 2.7% i n 2k hour t e s t s and found the f o l l o w i n g rank o r d e r i n g : 0.3% > .1% > .03% >.9% > 2.1%. In a more r e f i n e d t e s t we p a i r e d each c o n c e n t r a t i o n of .033$, 1.$, .2%, .3%, Λ% .5$, .6$, .7$, . 8 $ , .9%, and 1.0$ against a l l others i n a long term 2 b o t t l e t e s t . F i g . 3 shows the preference i n f e r r e d from these data. The short term t e s t s g i v e the opportunity t o look more c a r e ­ f u l l y at f a c t o r s which i n i t i a t e feeding and probably are more c l o s e l y r e l a t e d t o t a s t e and the immediate i n g e s t i o n consequences. An i n t e r e s t i n g example of the o v e r a l l d i f f e r e n c e between the short and l o n g term t e s t s has been the s t u d i e s of the r e l a t i v e accep­ t a b i l i t i e s of the sugars. In short term exposures the r a t accepts 9

In Flavor Chemistry of Animal Foods; Bullard, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

Methodology

SMITH AND RASHOTTE

of Behavioral

Testing

1 BOTTLE .2X1 > .5> .05> l.00> 2.00> .02> .01 2 BOTTLE 2.0>1.0>.5>.2>.1>.05>.02> .01

.01

.02

.05

.10

.20 .50

1.00 2.00

MOLAR CONCENTRATION OF SUCROSE Figure 1. Intake of sucrose (ml) plotted as a function of molar concentration for single-bottle, 24-hr tests. The solute (g) is also plotted as a function of molar concentration.

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f r u c t o s e > sucrose > glucose > maltose. I f long term t e s t s are used the r e l a t i v e acceptance i s reversed, i . e . , maltose > glucose > sucrose > f r u c t o s e . Systematic comparisons between s i n g l e b o t t l e and two b o t t l e procedures u s i n g short term t e s t s have not been made. One should, however, be very cautious i n g e n e r a l i z i n g from the s i n g l e b o t t l e t e s t t o the two b o t t l e t e s t even i f the t e s t i n g p e r i o d i s short. Quite o f t e n short term t e s t s i n v o l v e d e p r i v a t i o n schedules, which we have already seen a f f e c t p r e f e r ­ ences. H e s i t a t i o n i n e a t i n g and d r i n k i n g (neophobia) can be a problem i n short term t e s t s (15). F i n a l l y , i f the t e s t i s too short d i f f e r e n t r e s u l t s can occur. Beck et a l . , (h) have shown t h a t preference f o r sucrose drops from 90% t o 50% w i t h progres­ s i v e l y shorter i n t e r v a l s of repeated stimulus p r e s e n t a t i o n . Cohen and Tokieda (2k) and Cohen et_ a l . , (2£_) have shown t h a t water i s p r e f e r r e d over sucrose i f the two choice t e s t i s r e s t r i c t e d t o a few l i c k s . While the above d i s c u s s i o b o t t l e t e s t s , there are examples i n the l i t e r a t u r e of t e s t s w i t h m u l t i p l e foods (e.g., 26). In a d d i t i o n , procedures have been described where two b o t t l e s are a l t e r n a t e l y presented t o the animal i n a s e q u e n t i a l manner as f r e q u e n t l y as once a minute

(ι)· Most i n v e s t i g a t o r s make i n f e r e n c e s about the a c c e p t a b i l i t y of the food from the amount consumed. Knowledge of the patterns of e a t i n g can be q u i t e h e l p f u l i n making inferences about accept­ a b i l i t y . For example, i n studying the i n i t i a t i o n of e a t i n g both l a t e n c y t o b e g i n i n g e s t i o n and e a r l y r a t e of e a t i n g are important. We use s p e c i a l l i c k o m e t e r c i r c u i t s i n our i n v e s t i g a t i o n s of neo­ phobia i n the r a t and can make a "microscopic a n a l y s i s " of the f i r s t approach and h e s i t a n c y t o d r i n k a novel s o l u t i o n . In our s t u d i e s w i t h the glucose and s a c c h a r i n mixture we know t h a t from the f i r s t contact w i t h t h i s s o l u t i o n there i s a d i f f e r e n c e i n the o v e r a l l p a t t e r n of responding ( l l ) . W i t h i n two minutes, there i s a d i s t i n c t d i f f e r e n c e i n the p a t t e r n of l i c k i n g the mixture as compared t o l i c k i n g f o r glucose or s a c c h a r i n alone. In studying the maintenance of e a t i n g behavior one should know something of meal s i z e , e a t i n g responses t h a t occur without i n t e r r u p t i o n ( i . e . , the " e a t i n g b o u t " ) , the number of bouts per u n i t of time and the d i s t r i b u t i o n of i n t e r b o u t i n t e r v a l s . F i n a l ­ l y , i t i s important t o know the i n f l u e n c e of the b i o l o g i c a l c l o c k s ( d a i l y , e s t r u s , seasonal, etc.) on e a t i n g and d r i n k i n g behavior. Instrumental

Method

I t i s w e l l e s t a b l i s h e d t h a t s k e l e t a l responses are i n f l u e n c e d by t h e i r consequences. Responses which produce pleasant conse­ quences tend t o be repeated; those which produce a v e r s i v e con­ sequences tend t o be suppressed (27)· During the past 75 years p s y c h o b i o l o g i s t s have r e f i n e d t h i s simple a s s e r t i o n about the e f f e c t s of consequences on responses i n a program of research and

In Flavor Chemistry of Animal Foods; Bullard, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

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theory on the l e a r n i n g processes o f animals (28_, 29) » One s p i n o f f o f t h e i r work has been the development o f r a t h e r s o p h i s t i c a t e d l a b o r a t o r y procedures f o r studying the ways i n which d i f f e r e n t consequences i n f l u e n c e responding. A modern l a b o r a t o r y t r a i n i n g apparatus f o r the r a t i s shown s c h e m a t i c a l l y i n F i g u r e k. The apparatus i s a small enclosure w i t h a metal l e v e r prot r u d i n g from one w a l l , and an opening i n t o which s m a l l p e l l e t s of food can be dispensed. The l e v e r and the food dispenser i n t e r f a c e w i t h e l e c t r o n i c c i r c u i t r y which senses a l l l e v e r presses and dispenses food according t o a pre-arranged schedule. The schedules o f g r e a t e s t i n t e r e s t f o r our purposes are those i n which the l e v e r - p r e s s response i s " i n s t r u m e n t a l " i n producing food f o r the r a t . There are schedules i n which a l e v e r press must be made before food w i l l be dispensed. I n schedules o f t h i s sort we can study how the measureable aspects o f the l e v e r - p r e s s response (e.g., i t s frequency o animal exerts i n p r e s s i n the food obtained. I t should be understood t h a t the r a t / l e v e r press arrangement i s only one o f the many p o s s i b l e s p e c i e s / r e sponse arrangements which have been s t u d i e d w i t h the i n s t r u m e n t a l methodology. A sample o f the other common arrangements would i n c l u d e f i s h / d i s c - n o s i n g , b i r d s / d i s c - p e c k i n g , dogs/panel-pushing and monkeys/lever-pressing. I t t u r n s out t h a t l a w f u l f u n c t i o n s between the s t r e n g t h o f responding and the nature o f the consequence are r e a d i l y obtained i n devices o f the s o r t shown i n F i g u r e k provided t h a t c e r t a i n methodological precautions are observed. These f u n c t i o n s have encouraged many s c i e n t i s t s t o use the i n s t r u m e n t a l method as a supplement t o , o r i n p l a c e o f , the i n g e s t i o n a l method t o study a n i m a l s r e a c t i o n s t o v a r i o u s foods. For example, i t would be p o s s i b l e t o compare the s t r e n g t h o f the i n s t r u m e n t a l response when i t produces Food A w i t h t h a t when i t produces Food B. The l o g i c o f the i n s t r u m e n t a l method i s t h a t i f the foods are d i f f e r e n t i a l l y valued by the animal they should support d i f f e r e n t strengths of the i n s t r u m e n t a l response. But one might ask whether the i n s t r u m e n t a l methodology has anything s p e c i a l t o recommend i t . A f t e r a l l , the i n g e s t i o n a l method provides a p e r f e c t l y s t r a i g h t f o r w a r d way t o compare the a c c e p t a b i l i t y o f d i f f e r e n t foods, and the i n s t r u m e n t a l methodology simply measures a response one step removed from i n g e s t i o n to estimate the value an animal places on a given food. I n our o p i n i o n , the i n s t r u m e n t a l methodology provides a u s e f u l supplement t o the i n g e s t i o n a l method f o r i n v e s t i g a t i n g c e r t a i n aspects of the animal*s r e a c t i o n s t o foods. This method i s p a r t i c u l a r l y u s e f u l when i t i s a p p l i e d w i t h an understanding o f modern developments i n the i n s t r u m e n t a l technique. In the remainder o f t h i s paper we w i l l d i s c u s s two o f these developments i n some d e t a i l , i n c l u d i n g cautions which research i n d i c a t e s must be observed i n s u c c e s s f u l l y a p p l y i n g t h i s methodology. 9

1

In Flavor Chemistry of Animal Foods; Bullard, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

FLAVOR

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4>.3> 2> .5> .6> .1> .7>.8>.9>1.0> .033

.033 .1 .2 .3 .4 .5 .6 .7 .8 .9 1.0

SACCHARIN

CONCENTRATION

Figure 3. Saccharin concentrations from .03 to 1.0% were each paired with all other concentrations in 2-bottle, 24-hr tests. The percent of the pairings in which each of the concentrations was preferred is plotted.

Figure 4. Instrumental testing apparatus for the rat

In Flavor Chemistry of Animal Foods; Bullard, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

FOODS

3.

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Simple Reinforcement Schedules. A d i s t i n c t i v e c h a r a c t e r i s t i c of t h e i n s t r u m e n t a l methodology i s t h a t t h e " i n d i c a t o r " response which permits i n f e r e n c e s about the v a l u e o f a food i s performed independently o f the act o f i n g e s t i n g t h e food. I n t h e case o f the r a t i n F i g u r e U, the i n d i c a t o r response i s t h e l e v e r press which occurs a t a separate time and p l a c e from t h e e a t i n g o f t h e food p e l l e t . Because t h e i n d i c a t o r response and t h e i n g e s t i o n response are separate i n t h i s methodology, the i n v e s t i g a t o r has c o n s i d e r a b l e l a t i t u t e i n t a i l o r i n g some f e a t u r e s o f t h e t e s t s i t u a t i o n t o h i s needs. I n p a r t i c u l a r , t h e i n v e s t i g a t o r can r e a d i l y pace t h e animal's exposure t o t h e food d u r i n g a t e s t s e s s i o n , and can arrange f o r an extended t e s t s e s s i o n i n which the i n d i c a t o r response i s performed many t i m e s , perhaps even i n a s p e c i f i c way designed enhanc t h s e n s i t i v i t f tha sponse t o c e r t a i n aspect The means by which an i n v e s t i g a t o r gains l a t i t u d e i n a r ranging h i s t e s t s i t u a t i o n i s through t h e use o f schedules o f reinforcement which evolved from B. F. Skinner's e a r l y attempts to study t h e e a t i n g behavior o f r a t s (30, 31). The d i s t i n g u i s h i n g f e a t u r e o f a schedule o f reinforcement i s t h a t i t imposes a c r i t e r i o n which must be met before t h e animal's response can produce the food-consequence (32). We w i l l d i s c u s s t h e c r i t e r i a which c o n s t i t u t e t h e simple reinforcement schedules below, and then w i l l i l l u s t r a t e how these simple schedules can be put t o use i n e v a l u a t i n g foods. F i g u r e 5 i l l u s t r a t e s the c r i t e r i a employed i n t h e f o u r simp l e s t reinforcement schedules and t h e way i n which these c r i t e r i a i n f l u e n c e responding. There a r e f o u r b a s i c c r i t e r i a . When a " r a t i o " c r i t e r i o n i s employed the animal i s r e q u i r e d t o perform a s p e c i f i e d number o f responses (e.g., l e v e r presses) before t h e food-consequence i s produced. I n the f i x e d - r a t i o (FR) c e l l t h e c r i t e r i o n i s shown as t h e heavy l i n e i n t e r s e c t i n g the a x i s on which cumulative responses are p l o t t e d , but not i n t e r s e c t i n g t h e a x i s on which cumulative time s i n c e t h e l a s t f e e d i n g i s p l o t t e d . This means t h a t whenever t h e animal performs t h e number o f r e sponses r e q u i r e d by the c r i t e r i o n , i r r e s p e c t i v e o f how much time i t takes t o do so, the next response produces food. I n t h e v a r i a b l e - r a t i o (VR.) c e l l , the c r i t e r i o n v a r i e s u n p r e d i c t a b l y from one food p r e s e n t a t i o n t o t h e next (shown by t h e l i g h t l i n e s i n t e r s e c t i n g t h e response a x i s ) b u t , on the average, the c r i t e r i o n f a l l s a t the dark l i n e . A f t e r a moderate amount o f t r a i n i n g t h e b e h a v i o r a l e f f e c t s o f these schedules on responding between succ e s s i v e food p r e s e n t a t i o n s i s s t r i k i n g l y d i f f e r e n t . The hypot h e t i c a l response data i n t h e f i x e d - r a t i o c e l l show t h a t a f t e r a p e r i o d without responding immediately a f t e r a f e e d i n g , t h e r e i s an abrupt change t o a h i g h r a t e o f responding which i s maint a i n e d u n t i l t h e r e s p o n s e - c r i t e r i o n i s reached and food i s p r e sented again. The v a r i a b l e - r a t i o c e l l shows t h a t responding continues s t e a d i l y and a t a h i g h r a t e throughout t h e i n t e r f o o d interval.

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When an " i n t e r v a l " c r i t e r i o n i s employed a response w i l l produce food o n l y i f a designated p e r i o d o f time has elapsed s i n c e t h e l a s t food p r e s e n t a t i o n . A l l responses made "before t h a t c r i t e r i o n time have no programmed consequences. The f i x e d i a b l e - i n t e r v a l c e l l s o f the t a b l e i n F i g u r e 5 show t h a t when the c r i t e r i o n i n t e r v a l i s a f i x e d d u r a t i o n the animal does not respond immediately a f t e r a f e e d i n g , and then responds a t a g r a d u a l l y i n c r e a s i n g r a t e u n t i l the temporal c r i t e r i o n i s reached, whereupon the next response produces food. The c e l l l a b e l l e d "VI" shows t h a t when the c r i t e r i o n i n t e r v a l v a r i e s u n p r e d i c t a b l y around an average v a l u e , responding i s maintained a t a steady and moderate r a t e throughout t h e i n t e r v a l between f e e d i n g s . Consider some a p p l i c a t i o n s o f these reinforcement schedules i n the study o f foods. A p a r t i c u l a r l y u s e f u l f e a t u r e o f t h e i n t e r v a l schedules i s t h a t they a l l o w the i n v e s t i g a t o r t o space periods o f food i n g e s t i o out s a c r i f i c i n g a continuou As an extreme example, F e r s t e r & Skinner (32) t r a i n e d pigeons to peck a p l a s t i c d i s c on a V a r i a b l e I n t e r v a l 3-min schedule. This schedule allowed b r i e f access t o a mixed g r a i n r e i n f o r c e r f o l l o w i n g a peck-response a t u n p r e d i c t a b l e i n t e r v a l s whose average d u r a t i o n was 3-min. They r e p o r t e d t h a t t h e animals pecked at a r a t e o f about 1.5 pecks/sec throughout t e s t sessions which l a s t e d from 9 t o ik hours, o f t e n pecking more than 87,000 times per s e s s i o n t o o b t a i n only about 250 p r e s e n t a t i o n s o f g r a i n ! Because the i n v e s t i g a t o r can j o i n t l y v a r y the i n t e r v a l between food p r e s e n t a t i o n s and the volume o f food presented p e r reinforcement on f i x e d - i n t e r v a l schedules he can e a s i l y achieve c o n t r o l over t h e r a t e a t which the animal becomes s a t i a t e d d u r i n g an i n d i v i d u a l t e s t s e s s i o n . C o l l i e r & Myers (33) i l l u s t r a t e d how these f a c t o r s can i n t e r a c t i n s u b t l e ways t o i n f l u e n c e t h e i n d i c a t o r response. R e c a l l t h a t F i g u r e 3 showed t h a t i n a s i n g l e b o t t l e long-term t e s t r a t s d r i n k more o f a H i sucrose s o l u t i o n than o f a 2M s o l u t i o n . When C o l l i e r and Myers (33) used these same c o n c e n t r a t i o n s i n the i n s t r u m e n t a l methodology they found t h a t t h e r a n k i n g o f the two c o n c e n t r a t i o n s depend upon the schedule o f reinforcement used. The r a t s were t e s t e d i n 30-min d a i l y sessions o f l e v e r p r e s s i n g . When a F i x e d - I n t e r v a l 1-min schedule was used, t h e r a t s pressed the l e v e r more o f t e n when t h e r e i n f o r c i n g s o l u t i o n was 1M (32$ by weight) than when i t was 2M {6h%). However, t h e o p p o s i t e r e s u l t was obtained when a F i x e d I n t e r v a l U-min schedule was used: t h e r a t s pressed more o f t e n for t h e 2M s o l u t i o n than f o r the 1M s o l u t i o n . Thus, simply by lengthening the scheduled time between s u c c e s s i v e p r e s e n t a t i o n s of the sucrose s o l u t i o n s t h e schedules y i e l d e d an opposite ranking o f the two c o n c e n t r a t i o n s o f the s o l u t i o n . A d d i t i o n a l r e s e a r c h by C o l l i e r & Myers (33) i n d i c a t e d t h a t t h i s r e s u l t i s best understood as a simple consequence of t h e two schedules a l l o w i n g c e r t a i n p h y s i o l o g i c a l v a r i a b l e s t o a c t d i f f e r e n t i a l l y . They concluded t h a t l e v e r p r e s s i n g was d e t e r a n d

v a r

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mined by t h e j o i n t a c t i o n o f two independent processes, t a s t e ^- monetary s a t i a t i o n . Responding was d i r e c t l y r e l a t e d t o t a s t e ( i . e . , c o n c e n t r a t i o n ) when s a t i a t i o n was low, and t h e animals s a t i a t e d more q u i c k l y when t h e schedule allowed frequent access t o t h e s o l u t i o n s ( i . e . , F I 1 min.). A v a r i e t y o f evidence supported t h i s c o n c l u s i o n . For example, i n t h e e a r l y p a r t o f each d a i l y t e s t s e s s i o n (when s a t i a t i o n was n e c e s s a r i l y low) l e v e r p r e s s i n g was higher f o r t h e higher c o n c e n t r a t i o n i r r e s p e c t i v e o f t h e schedule employed. Furthermore, i n t h e l a t e r p a r t s of each t e s t s e s s i o n t h e r a t e o f l e v e r p r e s s i n g on t h e F I 1-min schedule d e c l i n e d , i m p l i c a t i n g a s a t i a t i o n e f f e c t . F i n a l l y , manipulation o f s a t i a t i o n by a d j u s t i n g t h e volume o f s o l u t i o n per reinforcement on t h e two schedules provided r e s u l t s c o n s i s t e n t w i t h t h e t a s t e / s a t i a t i o n account given above. The m a n i p u l a t i o n o f r e i n f o r c e r volume i n t h e C o l l i e r & Myers (32) experiment a l s o demonstrate b e h a v i o r a l v a r i a b l e s whic the i n s t r u m e n t a l methodology. That i s , some minimal volume o f reinforcement i s necessary f o r t h e i n s t r u m e n t a l response t o occur a t a l l . Larger minimal volumes are r e q u i r e d when t h e schedule arranges lengthy temporal spacings o f r e i n f o r c e r s . Our d i s c u s s i o n o f t h e C o l l i e r and Myers experiments i s i n t e n ded t o i l l u s t r a t e t h a t when foods are evaluated w i t h t h e i n s t r u mental methodology i t i s important t o c o n s i d e r t h e r o l e o f both the b e h a v i o r a l v a r i a b l e s (2ft,32) and t h e c o n f i g u r a t i o n o f phys i o l o g i c a l v a r i a b l e s which t h e v a r i o u s schedules o f reinforcement bring into play. Before c l o s i n g t h i s d i s c u s s i o n o f simple reinforcement schedu l e s we wish t o make a b r i e f comment on t h e r a t i o schedules. These schedules arrange a d i r e c t r e l a t i o n between t h e number o f i n s t r u m e n t a l responses made and t h e reinforcement, they have some obvious a p p l i c a t i o n s f o r e v a l u a t i n g foods. For example, u s i n g a single-response t e s t we c o u l d compare d i f f e r e n t foods by d e t e r mining how many responses an animal w i l l make t o o b t a i n each food. Beginning w i t h a low-valued f i x e d - r a t i o schedule such as FRIO (where food i s obtained a f t e r every 10th response), t h e c r i t e r i o n number o f responses f o r reinforcement would i n c r e a s e by, say, 5 responses a f t e r s u c c e s s f u l completion o f each r a t i o . I n t h i s incrementing f i x e d - r a t i o schedule we c o u l d then determine t h e "break-point" a t which t h e animal would r e f u s e t o make t h e c r i t e r i o n number o f responses t o o b t a i n t h a t food. Comparison of break-points f o r d i f f e r e n t foods would provide one estimate of t h e r e l a t i v e values o f the foods. One c a u t i o n i n u s i n g r a t i o schedules t o evaluate foods might be noted. On r a t i o schedules t h e i n d i v i d u a l responses tend t o become "chained" together so t h a t performance o f any one response i s determined by performance o f t h e immediately preceding r e sponse. Consequently, t h e r a t e o f response on t h e r a t i o schedu l e s may not be a s e n s i t i v e i n d i c a t o r o f the value o f a r e i n f o r c e r . However, other measures o f performance r e f l e c t a r e i n an

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f o r c e r * s e f f e c t i v e n e s s , p a r t i c u l a r l y the l e n g t h o f the pause a f t e r each r e i n f o r c e r (32). In the space a v a i l a b l e -we can o n l y draw a t t e n t i o n t o t h i s one nuance o f the d i f f e r e n t reinforcement schedules. Concurrent Reinforeement Schedules. Preference between foods i s s t u d i e d w i t h the i n g e s t i o n a l method by c o n f r o n t i n g the animal w i t h two foods (as i n the t w o - b o t t l e t e s t d i s c u s s e d earl i e r ) and computing the r a t i o of the amount consumed of one food to the t o t a l amount consumed. Such "preference r a t i o s " p r o v i d e the bases f o r i n f e r e n c e s about r e l a t i v e value o f the two foods. When the i n s t r u m e n t a l methodology i s employed i n preference t e s t i n g the animal i s confronted w i t h two response a l t e r n a t i v e s , as i n the apparatus shown f o r pigeons i n F i g u r e 6. In t h i s case, two p l a s t i c d i s c s are mounted on one w a l l of the t e s t chamber, above a hopper where foo the l e f t - h a n d d i s c migh d i s c Food B. The f i g u r e shows t h a t a preference r a t i o c o u l d be computed as the r a t i o of pecks on the l e f t d i s c t o the t o t a l number pecks on both d i s c s ( i . e . , L/L + R). In t h i s case a strong preference f o r the l e f t d i s c (and i t s a s s o c i a t e d food) would be expressed by a preference r a t i o near 1.0. While i t i s easy t o arrange an i n s t r u m e n t a l preference t e s t by r e q u i r i n g the animal t o peck once on e i t h e r d i s c t o o b t a i n the food a s s o c i a t e d w i t h t h a t d i s c , the p o t e n t i a l of the i n s t r u mental method i s most f u l l y r e a l i z e d when responding on the two d i s c s i s r e i n f o r c e d on the simple reinforcement schedules. Cons i d e r the w e l l - s t u d i e d case o f concurrent schedules i n which pecking on the l e f t and r i g h t d i s c s i s r e i n f o r c e d on separate and independent v a r i a b l e - i n t e r v a l schedules ( i . e . , cone VI VI schedule). F i g u r e 5 i l l u s t r a t e d t h a t VI schedules y i e l d c o n t i n uous responding at moderate r a t e s throughout the i n t e r v a l between food p r e s e n t a t i o n s , so t h a t on a cone VI VI schedule we can expect a steady stream of pecking throughout the t e s t s e s s i o n . The q u e s t i o n o f i n t e r e s t i s how the pigeon d i s t r i b u t e s i t s r e sponses on the two d i s c s as a f u n c t i o n of the p r o p e r t i e s of the foods a s s o c i a t e d w i t h each d i s c . F i g u r e 7 summarizes some aspects of concurrent VI VI schedu l e s which are important f o r the present d i s c u s s i o n . The top panel i l l u s t r a t e s t h a t when both schedules are i d e n t i c a l i n l e n g t h and both produce i d e n t i c a l foods the sequence of responses i s l i k e l y t o approach a simple a l t e r n a t i o n p a t t e r n . In f a c t , the animal can maximize the number of r e i n f o r c e r s i t o b t a i n s per u n i t time by a l t e r n a t e l y "checking" i n t h i s way whether the VI schedule on each d i s c i s ready t o p r o v i d e a r e i n f o r c e r . While t h i s i s an e f f i c i e n t way f o r the b i r d t o procède, and y i e l d s a preference r a t i o of approximately 0.5 (which would be expected i f , i n f a c t , the b i r d i s i n d i f f e r e n t t o the two i d e n t i c a l f o o d s ) , t h i s a l t e r n a t i n g p a t t e r n of responding proves not t o be o p t i m a l when foods w i t h d i f f e r e n t p r o p e r t i e s are compared. Consequently, i t i s

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INTERVAL

1

FIXED

1 /

I

/

/

—

CUMULATIVE TIME SINCE LAST FOOD PRESENTATION

VARIABLE

Figure 5. Criteria for making food available following a response on fixed- and variable ratio and interval schedules. Each cell of the table shows the criterion for that schedule and the typical pattern of responding in the interval between successive food presentations. The criterion is shown by the straight lines which intersect the response axis on the time axis. Responding is shown by the dotted line curve in each cell. The axes in each cell should be read as labeled in the fixed ratio cell.

Pecking discs Food Hopper

Apparatus for training pigeons to choose between food alternatives by pecking on discs. Preference

Ratio

_L L +R

Total pecks on left disc Total pecks on left + right disc

Figure 6. Instrumental test apparatus for studying preference of pigeons

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1. Hypothetical sequence of responses to left and right alternatives which produce the same food reinforcer ( no C O . D. imposed ) :

LRLRLRLLRLRRL 2. Hypothetical sequence of responses when C.O.D. imposed:

LLLRRRRLLRRRLLLLLRR 3. Hypothetica preference rati

(L/L+R)

Figure 7. Relevant features of concurrent schedules

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d e s i r a b l e t o e l i m i n a t e the a l t e r n a t i o n p a t t e r n o f responding hef o r e undertaking preference s t u d i e s w i t h the concurrent schedules methodology. An e f f e c t i v e method f o r "breaking up the a l t e r n a t i o n p a t t e r n i s t o impose a s o - c a l l e d "change-over-delay" (C.O.D.). Put simp l y , a C.O.D. p e n a l i z e s the animal f o r "changing over" from r e sponding on one d i s c t o responding on the other. The p e n a l t y i s i n the form o f a "delay" p e r i o d i n which pecks on one key are i n e f f e c t i v e i n producing any reward which might have "been schedu l e d t h e r e . This delay p e r i o d i s i n i t i a t e d by any peck on a d i s c which i s preceded by a peck on the other d i s c . The net r e s u l t o f t h i s d i a b o l i c a l scheme i s t h a t the pigeon can never obt a i n food by responding i n s t r i c t a l t e r n a t i o n from one key t o the other. Consequently, the pigeon changes i t s p a t t e r n o f responding and the i n v e s t i g a t o r achieves h i s purpose. The second panel o f F i g u r e 7 show t o peck s e v e r a l times o the other one. The importance o f the C.O.D. i s concurrent schedules becomes evident when we consider the bottom panel o f F i g u r e 7 which i l l u s t r a t e s a r e l a t i o n s h i p found when a C.O.D. i s employed. Each data p o i n t i n the f i g u r e i s h y p o t h e t i c a l , the f u n c t i o n shown i s r e p r e s e n t a t i v e o f a l a r g e number o f f i n d i n g s obtained w i t h concurrent VI V I schedules i n which a C.O.D. i s employed ( 3 M . Each data p o i n t represents a preference r a t i o which our hypot h e t i c a l pigeon would produce i n choosing between foods which d i f f e r i n q u a n t i t a t i v e o r q u a l i t a t i v e ways. For example, suppose the pigeon obtained the same type o f g r a i n r e i n f o r c e r by pecking on each d i s c , but a l a r g e r amount per r e i n f o r c e r f o r pecking on one o f the d i s c s . Suppose f u r t h e r t h a t the pigeon was t e s t e d w i t h v a r i o u s combinations o f d i f f e r ent amounts. I f f o r each combination we computed the r a t i o o f the amount obtained by pecking on the l e f t d i s c t o the t o t a l amount obtained by pecking both d i s c s , we would c a l c u l a t e the entity,

r

L

+

r

R

where r ^ and r r e p r e s e n t , r e s p e c t i v e l y , the t o t a l amounts o f food obtained by pecking the l e f t and r i g h t d i s c s i n a t e s t s e s s i o n . And, i f the preference r a t i o obtained when each comb i n a t i o n o f amounts was a v a i l a b l e were p l o t t e d a g a i n s t the r e i n forcement r a t i o c a l c u l a t e d f o r t h a t combination we would produce a graph very s i m i l a r t o t h a t shown i n the bottom panel o f F i g u r e 7· This graph i l l u s t r a t e s the most s t r i k i n g aspect o f the data produced by concurrent schedules: the value o f the preference r a t i o matches the value o f the reinforcement r a t i o . This funct i o n i s summarized as the s o - c a l l e d matching law which i s exR

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pressed as

L + R

r

L

+ r

R

where L and R designate t h e number o f responses t o t h e l e f t and r i g h t a l t e r n a t i v e s , r e s p e c t i v e l y , and r ^ and designate some measure o f t h e r e i n f o r c e r s a v a i l a b l e f o r responding on the two a l t e r n a t i v e s . The matching law was formulated by R. J . Hernns t e i n (35., 36, 31). There are f o u r p o i n t s t o be emphasized about t h e matching law. F i r s t , matching i s obtained more r e l i a b l y when a C.O.D. i s imposed. I t i s p r e s e n t l y unknown whether d i f f e r e n t lengths o f C.O.D. w i l l be r e q u i r e species (3k). Second r e i n f o r c e r s d i f f e r i n such p r o p e r t i e s as t h e i r amount, frequency of occurrence and q u a l i t y (3k). T h i r d , t h e r e have been s e v e r a l r e f o r m u l a t i o n s o f t h e matching law w i t h t h e g o a l i n mind of making i t more comprehensive (3k)· F i n a l l y , matching has been found i n a number o f v a r i a t i o n s on t h e cone VI V I procedure d i s cussed here but i t i s i m p o r t a n t l y i n f l u e n c e d by t h e parameters of t h e reinforcement schedules employed (3k_ 38)· D i s c u s s i o n of these developments i s beyond t h e scope o f t h i s paper. How might t h e d i s c o v e r y o f matching on concurrent schedules be u s e f u l i n e v a l u a t i n g animal foods? H. L. M i l l e r r e c e n t l y provided one example i n an experiment concerned w i t h s c a l i n g t h e hedonic v a l u e o f q u a l i t a t i v e l y d i f f e r e n t g r a i n s f o r pigeons (3ft)· Using a concurrent V I VI schedule on which pigeons pecked d i f f e r ent keys t o o b t a i n d i f f e r e n t g r a i n s , M i l l e r f i r s t e s t a b l i s h e d t h a t t h e pigeons p r e f e r r e d wheat t o buckwheat. Then, t h a t they p r e f e r r e d buckwheat t o hemp. He f i n a l l y p r e d i c t e d and demons t r a t e d t h a t t h e pigeons p r e f e r r e d wheat over hemp. The s p e c i a l f e a t u r e o f h i s work was t h a t through a mathematical a n a l y s i s o f h i s data i n s p i r e d by t h e matching law, M i l l e r was able t o cons t r u c t an e m p i r i c a l l y based s c a l e o f g r a i n q u a l i t y f o r t h e pigeon. His c a l c u l a t i o n s i n d i c a t e d t h a t i f buckwheat serves as the s t a n dard and i s a r b i t r a i l y assigned a v a l u e o f 10 q u a l i t y u n i t s , wheat would f a l l a t ik and hemp a t 9 q u a l i t y u n i t s . In developing animal foods i t would seem t o be o f some importance t o determine t h e r a n k i n g o f q u a l i t a t i v e l y d i f f e r e n t foods on a e m p i r i c a l l y based s c a l e . The concurrent schedules methodology provides one way o f a c h i e v i n g t h i s g o a l . I n f a c t , the concurrent schedules procedure seems p a r t i c u l a r l y w e l l s u i t e d f o r s c a l i n g q u a l i t a t i v e l y d i f f e r e n t foods because i t employs a common i n d i c a t o r response (e.g., key pecking) f o r both foods. In t h i s way these schedules l i t e r a l l y make i t f e a s i b l e t o undertake t h e p r o v e r b i a l comparison o f apples and oranges. Such comparisons are fraught w i t h d i f f i c u l t i e s when t h e i n g e s t i o n a l method i s used because q u a l i t a t i v e l y d i f f e r e n t foods o f t e n evoke 9

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g r e a t l y d i f f e r e n t i n g e s t i o n a l b e h a v i o r , thereby c o m p l i c a t i n g comparisons o f the foods. Conclusion In t h i s b r i e f review o f the p s y c h o b i o l o g i c a l approach t o food acceptance i n animals we have attempted t o provide both a warning and a temptation t o those who develop animal foods. While t h e r e i s nothing new i n the warning t h a t the v a r i a b l e s i n f l u e n c i n g food acceptance i n animals are complex, perhaps you have found something new i n our emphasis on the b e h a v i o r a l v a r i ­ ables i n food acceptance t e s t s . C e r t a i n l y , v a r i a b l e s such as t h e l e n g t h o f the t e s t p e r i o d , the number o f a l t e r n a t i v e foods a v a i l ­ a b l e , and the a n i m a l s past h i s t o r y are important determinants of i n g e s t i v e behavior. L i k e w i s e , i n the i n s t r u m e n t a l method, the type o f reinforcemen c u r r e n t l y a v a i l a b l e , an schedules procedures i m p o r t a n t l y i n f l u e n c e i n s t r u m e n t a l respond­ ing. Since one's i n f e r e n c e s about a food's a c c e p t a b i l i t y are determined both by the behavior we observe i n our t e s t s i t u a t i o n and our knowledge o f the v a r i a b l e s which c o n t r o l t h a t b e h a v i o r , i t seems important t h a t these b e h a v i o r a l v a r i a b l e s be given t h e i r due, along w i t h the p h y s i o l o g i c a l v a r i a b l e s , when t e s t s f o r food a c c e p t a b i l i t y are designed. With regard t o t e m p t a t i o n , we hope t h a t our l i m i t e d remarks w i l l tempt you t o b i t e more f u l l y i n t o the " f r u i t o f knowledge" which i s the l a r g e experimental and t h e o r e t i c a l l i t e r a t u r e on the p s y c h o b i o l o g i c a l approach t o food acceptance i n animals. 1

Literature Cited 1. Pfaffmann, C.; Fisher, G. and Frank, M. K. In "Olfaction and Taste II", T. Hayashi, Editor, 361-382, Pergamon Press, Oxford, (1976). 2. Young, P. T. Psychological Review, (1966) 73 59-86. 3. Horowitz, G. P.; Whitney, G.; Smith, J. C. and Stephan, F.K. Psychopharmacology (1977) 52 119-122. 4. Beck, R. C.; Nash, R.; Viernstein, L. and Gordon, L. Journal of Comparative and Physiological Psychology (1972) 78 40-50. 5. McLeary, R. A. Journal of Comparative and Physiological Psychology (1953)46411-421. 6. Puerto, Α.; Deutsch, J. Α.; Molina, F. and Roll, P. L. Science (1976) l92 485-487. 7. Valenstein, E. S. and Weber, M. L. Journal of Comparative and Physiological Psychology (1965)60443-446. 8. Kare, M. R. In "Olfaction and Taste III", Carl Pfaffmann, Editor, 586-592, Rockefeller University Press, N.Y. City, (1969). 9. Collier, G. and Bolles, R. Journal of Comparative and Phy­ siological Psychology (1968) 65 379-383.

In Flavor Chemistry of Animal Foods; Bullard, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

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10. Valenstein, E. S. ; Cox, J. W. and Kakolewski, J. W. Science (1967)157552-554. 11. Smith, J.C.;Williams, D. P. and Jue, S. S. Science (1976) 191 304-305. 12. DeWys, W. D. and Walters, K. Cancer (1975) 36 1888-1896. 13. Bradley, R. M. Doctoral Dissertation, Florida State Univer­ sity, Tallahassee, Florida 32306 (1970). 14. Barnett, S. A. "The Rat", Aldine Publishing Company, Chicago Ill. (1963). 15. Carroll, M. E.; Dine, H. I.; Levy, C. J. and Smith, J. C. Journal of Comparative and Physiological Psychology (1975) 82 457-467. 16. Garcia, J.; Kimeldorf, D. J. and Koelling, R. A. Science (1955) 122 157-158. 17. Gamzu, E. In "Learning Mechanisms in Food Selection" Barker L. M.; Best, M. R University Press, , (1977) 18. Young, P. T. Psychological Review (1968) 75 222-241. 19. Stellar, E. and McCleary, R. A. American Psychologist (1952) 7 256. 20. Collier, G. and Bolles, R. Journal of Comparative and Physi­ ological Psychology (1968) 65 379-383. 21. Richter, C. P. and Campbell, Κ. Ε. H. Journal of Nutrition (1940) 20 31-46. 22. Hagstrom E. C. and Pfaffmann, C. Journal of Comparative and Physiological Psychology (1959) 52 259-262. 23. Young, P. T. and Greene, J. T. Journal of Comparative and Physiological Psychology (1953) 46 288-29524. Cohen, P. S. and Tokieda, F. K. Journal of Comparative and Physiological Psychology (1972) 79 254-258. 25. Cohen, P. S.; Wier, J. and Granat, M. B. Physiology and Behavior (1975)14383-386. 26. Fuller, J. L. Journal of Comparative and Physiological Psy­ chology (1964) 57 85-88. 27. Thorndike, E. L. Psychological Monographs (1898) 2 (4, Whole No. 8). 28. Kimble, G. A. "Hilgard and Marquis' Conditioning and Learn­ ing 2 Edition", Appleton-Century-Crofts, New York (1961). 29. Mackintosh, N. J. "The Psychology of Animal Learning". Academic Press, London (1974)· 30. Skinner, B. F. In "Psychology: A Study of a Science, Vol. 2" S. Koch, Editor, 359-379, McGraw-Hill, New York (1959). 31. Collier, G.; Hirsch, E.; and Kanarek, R. In "Handbook of Operant Behavior". W. K. Honig and J. E. R. Staddon, Editors, 28-52 Prentice-Hall Inc., Englewood Cliffs, N.J. (1977). 32. Ferster, C. B. and Skinner, B. F. "Schedules of Reinforce­ ment". Appleton-Century-Crofts, New York (1957). 33. Collier, G. and Myers, L. Journal of Experimental Psychology (1961)6157-66.

In Flavor Chemistry of Animal Foods; Bullard, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

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Methodology of Behavioral Testing

34. deVilliers, P. In "Handbook of Operant Behavior", W. K. Honig and J. E. R. Staddon, Editors, 233-287, Prentice-Hall Inc., Englewood Cliffs, N. J. (1977). 35· Herrnstein, R. J. Journal of the Experimental Analysis of Behavior (1961) 4 243-266. 36. Herrnstein, R. J. Journal of the Experimental Analysis of Behavior (1970) 13 243-266. 37. Rachlin, H. "Behavior and Learning", W. H. Freeman & Co., San Francisco (1976). 38. Fantino, E. and Navarick D. In "The Psychology of Learning and Motivation, Vol. 8". G. H. Bower, Editor, 147-185 Academic Press, N. Y. (1974). 39· Miller, H. L., Jr. Journal of the Experimental Analysis of Behavior (1976) 26,335-347. RECEIVED October 25, 1977.

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4 Isolation, Identification, and Biological Activity Assay of Chemical Fractions from Estrus Urine Attractive to the Coyote E. L. MURPHY, ROBERT A. FLATH, DALE R. BLACK, THOMAS R. MON, and ROY TERANISHI Western Regional Research Laboratory, Agricultural Research Service, U. S. Department of Agriculture, Berkeley, CA 94710 R. M. TIMM and W. E. HOWARD University of California, Davis CA 95616 Most of the sheep are by coyotes. Ranchers or farmers who have recently quit the business of raising sheep most often blame the lack of control of coyotes as being the precipitating cause for their decision. The greatest problem of using a device or chemical to affect coyote populations is to get the attention of the coyote. Excessive numbers of chemical baits must be dropped or a great number of "Coyote Getters" must be set to reach one coyote. Treating with an effective attractant would significantly reduce the cost and work involved. Further, it is very desirable that the attractant be specific for coyotes to reduce the injury and death to non­ -target species such as skunks, bobcats, eagles and pet dogs. Many attempts have been made to concoct brews or mixtures to be irresistably attractive for the coyote as he passes a dropped bait or trap. Mixtures of blood, animal organs and urine brewed or fermented in numerous ways have been used for years by ranchers and trappers with varying success. One ingredient common to many scent bait mixtures has been coyote urine. Several researchers have noted that significantly more coyotes have been caught with estrus than with non-estrus urine. This paper reports the initial efforts to isolate and identify an attractant from estrus urine which might prove specific for the coyote. Experimental Estrus Urine Collection. Female coyotes were confined singly in small cages with wire bottoms below which was fitted a stainless collection tray which sloped to empty into a one pint glass canning jar. In order to reduce fecal and drinking water contamination of the collected urine, the coyotes were fed in the morning and in the afternoon all food scraps and water were removed and the cages and collection trays cleaned. The jars were then put in place and urine collected overnight. In the morning the jars of urine were collected, frozen and stored at -20°C. The coyotes were then fed and the collection cycle repeated. For the purposes © 0-8412-0404-7/78/47-067-066$05.00/0 In Flavor Chemistry of Animal Foods; Bullard, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

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of t h i s study u r i n e was considered estrus urine when c o l l e c t e d i n a period between the appearance and disappearance of vaginal bleeding. Chemical F r a c t i o n a t i o n . The frozen urine was thawed and pooled in one 50 £ batch f o r chemical f r a c t i o n a t i o n . It was made basic (pH 12) with potassium hydroxide and the basic and neutral compounds extracted with d i e t h y l ether according to the e x t r a c ­ t i o n scheme o u t l i n e d i n Figure 1. The gel f r a c t i o n separated from the coyote urine a f t e r i t had been made b a s i c and was recov­ ered by f i l t r a t i o n of the basic u r i n e . The b a s i c compounds were separated by e x t r a c t i o n with 6N h y d r o c h l o r i c a c i d leaving the neu­ t r a l compounds i n the ether r e s i d u e . The o r i g i n a l ether e x t r a c ­ ted b a s i c u r i n e was then made a c i d (pH 2) and the a c i d i c com­ pounds extracted with d i e t h y l ether The a c i d i c basic and neu t r a l f r a c t i o n s were s t r i p p e Bioassay of Urine F r a c t i o n s . The degree of a t t r a c t i o n of the estrus u r i n e f r a c t i o n s , a c i d i c , b a s i c , neutral and urine r e s i ­ due, was estimated by presenting each f r a c t i o n with i t s s o l v e n t c o n t r o l in a randomized sequence to each of s i x coyotes i n r e p l i ­ cated t r i a l s . Each coyote was r e l e a s e d f o r t h i r t y minutes i n t o a large pen, 6m χ 20m, Figure 2, containing the t e s t urine f r a c t i o n with i t s solvent control i n separate portable scent s t a t i o n s on the ground and i t s behavior recorded by a hidden observer as i t approached each scent s t a t i o n ( l j . Time at each scent s t a t i o n as well as behavior such as r u b - r o l l i n g , chewing, s n i f f i n g , s c r a t c h i n g and scent marking was recorded by the observer. Three male and three female coyotes were used. Component I d e n t i f i c a t i o n . In preparation f o r gas chromato­ graphy the a c i d i c urine f r a c t i o n was reacted with diazomethane produced from DIAZALD, N-methyl-N-nitroso-p-toluenesulfonamide, A l d r i c h Chemical Company (2). As the diazomethane was generated by the a d d i t i o n of potassium hydroxide, the ethereal diazomethane s o l u t i o n was d i s t i l l e d i n t o an i c e bath-cooled r e a c t i o n f l a s k which contained the urine acids f r a c t i o n . The production of diazomethane was continued u n t i l a b r i g h t yellow c o l o r p e r s i s t e d i n the r e a c t i o n f l a s k . The r e a c t i o n mixture was allowed to s i t i n a hood overnight during which time the i c e melted allow­ ing the r e a c t i o n mixture to come to room temperature ( 2 3 ° C ) . The ether solvent was then removed by d i s t i l l a t i o n on a steam bath. P r e l i m i n a r y gas chromatographic runs were made on a Hewlett/Packard, Model 5831 A, gas chromatograph. Separation of the methyl e s t e r i f i e d components i n the a c i d i c u r i n e f r a c t i o n was made on a 0.03 i n . i . d . X500 f t s t a i n l e s s s t e e l open tubular c o l ­ umn coated with methyl s i l i c o n e o i l , SF 96-50 (General E l e c t r i c ) containing 5% Igepal CO-880 (General A n i l i n e and Film) using a flame i o n i z a t i o n d e t e c t o r . A f t e r sample i n j e c t i o n , the columns were held at 76°C f o r 30 min; then the oven tenperature was

In Flavor Chemistry of Animal Foods; Bullard, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

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1. BASE 2 ETHER INITIAL ETHER EXTRACT

BASIC EXTRACT

ACID

ACIDIC EXTRACT

ETHER EXTRACT

(NEUTRALS)

ETHER EXTRACT

(BASES)

ETHER EXTRACT

(ACIDS)

URINE RESIDUE

(RESIDUE)

AQUEOU EXTRACT

Figure 1. Urine fractionation procedure

Figure 2. Coyote test area

In Flavor Chemistry of Animal Foods; Bullard, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

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programmed at 2°/min to 210°C. T e n t a t i v e i d e n t i f i c a t i o n s were made from mass s p e c t r a l data obtained by coupling the e f f l u e n t end of the gas chromatograph column to a quadrupole-type mass spectrometer through a s i n g l e stage s i l i c o n e membrane i n t e r f a c e (Quad 300, E l e c t r o n i c Associates Inc.) as described by F l a t h , Forrey and Guadagni (3), and F l a t h and Forrey (4). Results and Discussion An e r r o r which i s d i f f i c u l t to avoid i n the c o l l e c t i o n method described i s the i n t r o d u c t i o n of a r t i f a c t chemical compounds i n t o the urine during c o l l e c t i o n which show up in the l a t e r chemical analysis. Contamination of the c o l l e c t e d urine by h a i r , f e c e s , d r i n k i n g water, food s c r a p s metabolic byproducts of m i c r o b i a l fermentation and a i r o x i d a t i o c o l l e c t i o n procedure. T t i o n f u r t h e r , u r i n e can be c o l l e c t e d under a t h i n l a y e r of toluene in a r e s e r v o i r with continuous urine flow i n t o a r e f r i g e r a t e d c o l l e c t i o n j a r where i t i s r a p i d l y f r o z e n . Experience with human urine c o l l e c t i o n i n d i c a t e d l i t t l e change in chemical composition of frozen urine a f t e r several months storage. When whole undiluted coyote urine i s gas chromatographed on high r e s o l u t i o n c a p i l l a r y columns with s o p h i s t i c a t e d computer comparison of the recorder t r a c i n g s , no s i g n i f i c a n t d i f f e r e n c e s can be demonstrated between e s t r u s and non-estrus u r i n e . Theref o r e , there must be an extensive reduction in the number of chemi c a l compounds by p r e f r a c t i o n a t i o n before i d e n t i f i c a t i o n of the i n d i v i d u a l chemical components can be attempted. One of the o l d e s t and most common approaches f o r the i s o l a t i o n and i d e n t i f i c a t i o n of the chemical cause of an a c t i v i t y i s the step by step chemical or physical separation of a natural products mixture while f o l l o w i n g the a c t i v i t y by an e f f e c t i v e bioassay. At each step the chemist must wait before proceeding with the next separat i o n f o r the r e s u l t s of the bioassay (often conducted by other members of a m u l t i - d i s c i p l i n e s c i e n t i f i c team) to i d e n t i f y the chemical f r a c t i o n which contains the major p o r t i o n of the a c t i v i t y . The chemical s e p a r a t i o n , Figure 1, produced four coyote urine f r a c t i o n s f o r t e s t i n g : a c i d s , bases, n e u t r a l s and urine r e s i d u e . The design and operation of a s u i t a b l e assay f o r the measurement of b i o l o g i c a l a c t i v i t y almost always poses the greater problem f o r the chemist r a t h e r than the various chemical separations. Because i t i s a part of his t r a i n i n g and he has the necessary s p e c i a l i z e d equipment, the chemist should p a r t i c i p a t e in the prepa r a t i o n of the t e s t samples. He can advise as to choice of s o l vents, exact c o n c e n t r a t i o n s , v o l a t i l i t y in transport and p l a c e ment, s t a b i l i t y to heat and oxygen of the a i r as well as in poss i b l e sources of contamination. But at the t e s t pen, using t e s t animals as v a r i a b l e and complex as the coyote, the chemist gives way to the s p e c i a l i s t in w i l d l i f e biology as to animal care and

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handling, t e s t design, accurate and o b j e c t i v e observation and most important, the i n t e r p r e t a t i o n of behavior. Figures 3 and 4 (_5) i l l u s t r a t e that of the four t e s t f r a c t i o n s , the acids f r a c t i o n e l i c i t e d the most time spent by the coyotes i n s n i f f i n g or r u b - r o l l i n g behavior. With c o n s i d e r a t i o n as to both t o t a l behavioral events, Figure 5, and time spent at the t e s t odor, Figure 6, a l l coyotes were a t t r a c t e d more by the acids f r a c tion. Comparison of r u b - r o l l i n g behavior of the acids f r a c t i o n with the urine r e s i d u e , which i s almost background, i n d i c a t e s that the e x t r a c t i o n procedure removed most of the compounds r e s p o n s i b l e f o r t h i s behavior. It i s i n t e r e s t i n g that the acids f r a c t i o n exh i b i t s greater r u b - r o l l i n g s t i m u l a t i o n than the commercially a v a i l a b l e coyote trap scent. Both bases and neutrals f r a c t i o n s were lower in s n i f f i n g or r u b - r o l l i n g response. Scent marking (Figure 7) and scraping (Figure 8) behavior was demonstrated by the coyotes longer in respons c l e a r l y a d i f f e r e n t behavio i s d i f f i c u l t to e x p l a i n , other than that t h i s f r a c t i o n may contain one or more chemicals a s s o c i a t e d with t e r r i t o r i a l marking. In a l l cases, the coyotes paid very l i t t l e more a t t e n t i o n to the gel f r a c t i o n than to the ether c o n t r o l or urine r e s i d u e . Pen t e s t s have several disadvantages f o r t e s t i n g u r i n e . The close r e s t r a i n t of even a large observation pen and the necessary occasional movement of personnel undoubtedly produce d i f f e r e n c e s in the behavior of penned coyotes as compared to the animal i n i t s natural h a b i t a t . F u r t h e r , pens tend to become u r i n e saturated from scent marking from previous t e s t i n g such that the urine f r a c t i o n s are being t e s t e d against a high background "noise l e v e l " . Again t e s t design may be used to compensate f o r some of the d i f f i culties. The t e s t coyotes should be housed and tested i n a remote area f r e e from excessive v e h i c l e and human t r a f f i c . Some coyotes l i k e some people are anosmic; t h e r e f o r e , only animals should be chosen f o r t e s t i n g which r e a d i l y respond to some standard odor of proven a t t r a c t a n c y and f a i l to demonstrate s i g n i f i c a n t i n t e r e s t in a blank t e s t sample of low odor content. Test s t a t i o n s or l o c a t i o n s of t e s t samples should be moved about i n the pen. T e s t samples should be presented i n a randomized sequence and r e p l i cated with as many animals as permitted by the resources and t e s t o b j e c t i v e s of the experiment. The chromatogram by g a s - l i q u i d chromatography, Figure 9, i n d i c a t e d over t h i r t y major peaks or chemical compounds i n the acids f r a c t i o n of the estrus coyote u r i n e . Mass s p e c t r a l data permitted t e n t a t i v e i d e n t i f i c a t i o n of the methyl e s t e r s of a s e r i e s of short chain f a t t y a c i d s , CO-C-.Q, together with aromatic compounds as present. Table 1 l i s t s 19 t e n t a t i v e i d e n t i f i c a t i o n s of compounds i n the acids f r a c t i o n . Because other i n v e s t i g a t o r s {6) have reported that an a r t i f i c i a l mixture of s i m i l a r f a t t y acids demonstrated s i g n i f i c a n t a t t r a c t i o n of coyotes i t w i l l be i n t e r e s t i n g to prepare a mixture of the f a t t y acids i d e n t i f i e d i n coyote u r i n e 1n the exact r a t i o that they are 1n

In Flavor Chemistry of Animal Foods; Bullard, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

MURPHY

E TAL.

Chemical

Fractions

from

Estrus

Urine

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