Flavor Chemistry of Animal Foods

Flavor Chemistry of Animal Foods Roger W. Bullard, EDITOR U.S. Fish and Wildlife Service A symposium sponsored by the D...

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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


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.


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


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


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 .



- 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 ,


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



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.


16

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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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Animal Flavor Research

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


18


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.


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


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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


24

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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


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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.


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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


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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


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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 . ,


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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


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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


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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


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.


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


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 Nôctylamine 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

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


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


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


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 . ,


FLAVOR

38

CHEMISTRY

O F ANIMAL

FOODS

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,


2. SHUMAKE


39

Univ. Chicago Press, Chicago, 1961. 10. Nachman, M. and Hartley, P. L., J. Comp. Physiol. Psychol. (1975) 89:1010-1018. 11. Sheffield F. D. and Roby, T. B. J. Comp. Physiol. Psychol. (1950) 43:471-481. 12. Kennedy, J. M. and Baldwin, B. A. Anim. Behav. (1972) 20: 706-718. 13. Rogers, J.G.,Jr. J. Wildl. Manage. (1974) 38:418-423. 14. Sterner, R. T. and Shumake, S. A. "Coyotes: Biology, Behavior, and Management," Bekoff, M., Ed., pp. 297-325, Academic Press, New York (in press). 15. Warren, R. P. and Pfaffmann, C. J. Comp. Physiol. Psychol. (1959) 52:263-266. 16. Arnold, G. W. and Hill, J. L. "Phytochemistry and Ecology: Proceedings of the Phytochemical Society Symposium," Harborne J. B., Ed., pp. 71-101 17. Young, P. T. Psychol (1966) 18. Thompson, R. D., Shumake, S. A. and Bullard, R. W. Proc. Fifth Vert. Pest Conf. (1972) 5:36-42. 19. Pager, J. Physiol. Behav. (1974) 12:189-195. 20. Garcia, J., Kover, R. and Green, K. F. Psychon. Sci. (1970) 20:313-314. 21. Clark, M. M. and Galef, Jr., B. G. Anim. Behav. (1977) 25:298-316. 22. Eibl-Eikesfeldt, I. "Ethology, the Biology of Behavior," Holt, Rinehart, and Winston, New York, 1970. 23. Rozin, P. Life Sci. Res. Rep. (1976) 2:285-312. 24. Young, P. T. "Handbook of Physiology, Sect. 6: Alimentary Canal," I, Code, C. and Heidel, W., Eds., pp. 353-366, American Physiological Society, Washington, 1967. 25. Kare, M. R. and Ficken, M. S. "Olfaction and Taste," Zotterman, Y., Ed., pp. 285-297, Macmillan, New York, 1963. 26. Morrison, G. R. and Young, J. C. Physiol. Behav. (1972) 8:29-32. 27. Nachman, M. and Cole, L. P. "Handbook of Sensory Physiology," IV, Beidler, L. M., Ed., pp. 337-362, Springer-Verlag, Berlin, 1971. 28. Campbell, C. S. and Davis, J. D. Physiol. Behav. (1974) 22:357-365. 29. Maller,O.,Kare, M. R., Welt, M. and Behrman, H. Nature (1967) 213:713-714. 30. Pilcher, C. W. T. and Jarman, S. P. J. Com. Physiol. Psychol. (1974) 87:56-61. 31. Contreras, R. J. and Hatton, G. I. Physiol. Behav. (1975) 15:569-576. 32. Rozin, P. J. Comp. Physiol. Psychol. (1965) 59:98-101. 33. Rozin, P. and Kalat, J. W. Psychol. Rev.(1971)78:459-486. 34. Pain, J. F. and Booth, D. A. Psychon. Sci. (1968) 10:363364. 35. Scott, Ε. M. and Quint, E. J. Nutr. (1946) 32:113-119.


40


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.


2. SHUMAKE 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83. 84. 85. 86. 87. 88. 89. 90. 91. 92. 93. 94. 95. 96. 97.


41

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:


42


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.


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.

44

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CHEMISTRY

OF

ANIMAL

FOODS

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


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


46

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CHEMISTRY

OF

ANIMAL

FOODS

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


3.

SMITH

AND RASHOTTE

Methodology

of Behavioral

47

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


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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


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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

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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

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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


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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

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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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Urine

67

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


FLAVOR

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COYOTE URINE

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


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69

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


MURPHY

E TAL.

Chemical

Fractions

from

Estrus

Urine

6 C O Y O T E S IN 48 INDIVIDUAL TESTS

" c ο S

so 60

40

^

h -

20

—J

i/i en i/i

LU

Q

ÛÉ

LU _ l

LU

° Siπ < 2

100 h~ 6 C O Y O T E S IN 48 INDIVIDUAL TESTS

c ο

S

80 1-

60

10^ organisms/ml (b) L a c t o b a c i l l i were sought i n 37 samples from 15 foxes o n l y . L a c t o b a c i l l i were only detected i n foxes l e s s than 3 months o l d (4 o u t o f 6 foxes i n t h i s age group) Fox a n a l sac s e c r e t i o n samples were a l s o examined anaerob i c a l l y using pre-reduced, a n a e r o b i c a l l y s t e r i l i z e d media (32), t a k i n g r i g i d p r e c a u t i o n s t o avoid exposure t o the a i r . Anaerobes were c l a s s i f i e d by o b t a i n i n g API 20A biochemical p r o f i l e s (Analytab Products Inc.) and employing s i n g l e l i n k a g e c l u s t e r a n a l y s i s (33) t o group s i m i l a r organisms w i t h s e l e c t e d API 20A reference species (31). Some 127 d i f f e r e n t anaerobic i s o l a t e s from 66 s e c r e t i o n s from 18 foxes y i e l d e d predominantly C l o s t r i d i a (Table I I , column A ) . An examination o f 107 f u r t h e r i s o l a t e s from 37 s e c r e t i o n s from 6 foxes, c o l l e c t e d under the most r i g o r o u s anaerobic c o n d i t i o n s and employing a s p e c i a l l y designed c o n t a i n e r to exclude a i r and maintain a carbon d i o x i d e environment around


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the hind-quarters o f the sedated f o x during sample c o l l e c t i o n , revealed the presence o f o t h e r , more oxygen-sensitive anaerobes (Table I I , column B ) . Anaerobic p o p u l a t i o n determinations are s u b j e c t t o e r r o r because o f the u n c e r t a i n t y o f percentage r e c o v e r a b i l i t y i n primary c u l t u r e . From microscopic counts, s t r i c t anaerobic p o p u l a t i o n s were estimated t o be comparable w i t h those o f aerobes, f r e q u e n t l y i n the range o f ΙΟ^-ΙΟ organisms/ ml s e c r e t i o n . 10

Table I I . Occurrence o f s t r i c t anaerobes i n r e d f o x anal sac s e c r e t i o n s (31). I s o l a t e s c l u s t e r i n g a t s i m i l a r i t y c o e f f i c i e n t 0.75 on the b a s i s o f API 20A biochemical prο f i l e s, morphology and Gram s t a i n r e a c t i o n (see t e x t ) Reference organism

A ^

B ^

Clostridium perfringen C l o s t r i d i u m ramosum C l o s t r i d i u m sporogenes C l o s t r i d i u m bifermentans Eubacterium lentum Bifidobacterium e r i k s o n i i Fusobacterium nucleatum Fusobacterium negrogenes Bacteroides f r a g i l i s f r a g i l i s Peptostreptococcug anaerobius unclustered

16 27 21 13 0 0 0 0 0 0

0 4 1 4 2 6 17 20 7 (O 9

(a) From 66 s e c r e t i o n s from 18 foxes, employing anaerobic techniques. (b) From 37 s e c r e t i o n s from 6 foxes, employing anaerobic techniques i n c l u d i n g an a i r - f r e e sampling environment, see text. (c) C l u s t e r i n g a t lower s i m i l a r i t y w i t h B i f i d o b a c t e r i u m e r i k s o n i i (6) and Peptostreptococcus anaerobius ( 3 ) . P r e l i m i n a r y observations i n d i c a t e t h a t the f o x anal sac may generate h i g h l y reducing c o n d i t i o n s , w i t h redox p o t e n t i a l s down to -400mV. A redox p o t e n t i a l o f -200mV has been recorded i n the cecum o f the mouse (34). I n the a n a l sac, i t appears t h a t f a c u l t a t i v e anaerobic organisms, such as s t r e p t o c o c c i and Proteus spp. create and maintain an anaerobic environment i n which s t r i c t anaerobes ( f r e q u e n t l y a c t i v e odor producers) can grow. In accord w i t h t h i s , chemical s t u d i e s so f a r undertaken on the a n a l sac s e c r e t i o n s o f the r e d f o x and other c a r n i v o r e s i n d i c a t e t h a t major low molecular weight components present are g e n e r a l l y commonly encountered products o f m i c r o b i a l a c t i v i t y . V o l a t i l e f a t t y a c i d s are major c o n s t i t u e n t s o f the a n a l sac s e c r e t i o n s o f the r e d f o x and the l i o n (29,35), the coyote (36),


5.

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Action

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Signalling

83

the domestic dog (36), and the mink (37). They are a l s o present i n the anal pocket s e c r e t i o n of the Indian mongoose (38) and i n the p e r i n e a l gland s e c r e t i o n o f the guinea p i g (39). In the cases of the red fox and of the Indian mongoose, evidence has been advanced to confirm t h e i r m i c r o b i a l o r i g i n . These same a c i d s occur i n a v a r i e t y of other mammalian scent sources, i n c l u d i n g the vagina o f primates, where a m i c r o b i a l o r i g i n has a l s o been s u b s t a n t i a t e d and a communicatory r o l e discussed (40), as w e l l as i n the v a g i n a l f l u i d s of other s p e c i e s , such as mink (41). Thus, i n the case of fox anal sac s e c r e t i o n , an inoculum of f r e s h anal sac s e c r e t i o n (100 μΐ) c o l l e c t e d a n a e r o b i c a l l y and incubated a n a e r o b i c a l l y i n pre-reduced Robertson's cooked meat medium (10 ml) a t 37 C f o r 48 hours y i e l d e d v o l a t i l e f a t t y acids a t concentrations ( a c e t i c a c i d , 170 mM; t o t a l C^-Cg a c i d s , 95 mM, by gas chromatography) comparable with, or i n excess of, those encountered i n anal sa b a c t e r i a l i s o l a t e s fro examined f o r v o l a t i l e f a t t y a c i d production under s i m i l a r c o n d i t i o n s . Anaerobes of a l l genera produced some or a l l the v o l a t i l e f a t t y a c i d s noted i n anal sac s e c r e t i o n , c e r t a i n C l o s t r i d i a and e u b a c t e r i a being the most a c t i v e producers, although great v a r i a t i o n s i n production were noted between d i f f e r e n t i s o l a t e s from the same genus. In the sac i t s e l f , m i c r o b i a l production of v o l a t i l e f a t t y a c i d s was confirmed by i r r i g a t i n g the sac with 1% aqueous sodium h y p o c h l o r i t e (containing 16.5% sodium c h l o r i d e ) , followed with p h y s i o l o g i c a l s a l i n e and then by f i l l i n g the sac with a n t i b i o t i c (10% a m p i c i l l i n / 0 . 5 % t e t r a c y c l i n i n p h y s i o l o g i c a l s a l i n e ) . Neither b a c t e r i a nor v o l a t i l e f a t t y a c i d s were detected i n the sac f o r i n excess o f 3 days subsequently. Other anal sac c o n s t i t u e n t s which are commonly encountered products of m i c r o b i a l a c t i v i t y i n c l u d e trimethylamine, noted i n the anal sac s e c r e t i o n s of the red fox (42), coyote and domestic dog (36), and the aromatic a c i d s p h e n y l a c e t i c a c i d and 3-phenylp r o p i o n i c a c i d (and r e l a t e d phenolic a c i d s ) , together with the diamines p u t r e s c i n e and cadaverine as w e l l as ammonia i n the anal sac s e c r e t i o n s of the red fox and the l i o n (25,29,35). Indole has a l s o been noted. The lower molecular weight l i p i d s o f l i o n anal sac s e c r e t i o n i n c l u d e many substances expected as h y d r o l y s i s products of sebaceous l i p i d s (25) . Red fox anal sac s e c r e t i o n a l s o e x h i b i t s an anomalous f r e e amino-acid composition with 5-aminovaleric a c i d predominating (43). The p o s s i b i l i t y that the s u l f u r - c o n t a i n i n g v o l a t i l e s present i n mustelid anal sac secre t i o n s are of m i c r o b i a l o r i g i n i s a t present under i n v e s t i g a t i o n i n our l a b o r a t o r i e s . f

Fermentative Scent Sources: II) Environmental;

Carrion

C a r r i o n , which may be detected a t l e a s t i n p a r t by odor cues, possesses considerable a t t r a c t i o n f o r c a r n i v o r e s , whether as a


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food source or as a s i t e f o r scent marking. M i c r o b i a l p u t r e f a c t i o n a r i s e s i n the carcass of a dead animal l a r g e l y as the r e s u l t o f b a c t e r i a l p e n e t r a t i o n of the gut a f t e r r e s i d u a l t i s s u e a n t i m i c r o b i a l a c t i v i t y has been l o s t and when anoxic c o n d i t i o n s have been e s t a b l i s h e d as the r e s u l t o f continued t i s s u e metabo l i s m i n the absence of oxygen. S t u d i e s i n t h i s area are few and have been undertaken p r i n c i p a l l y i n the context o f f o r e n s i c s c i e n c e . A v a l u a b l e review o f t h i s work i s p r o v i d e d by Corry (44), who r e p o r t s t h a t , although many d i f f e r e n t anaerobes r e s i d e i n the i n t e s t i n e , o n l y a few groups have been i m p l i c a t e d so f a r as major c o l o n i z e r s of corpses d u r i n g p u t r e f a c t i o n . The most important o f these i s C l o s t r i d i u m p e r f r i n g e n s , a vigorous s a c c h a r o l y t i c , l i p o l y t i c and p r o t e o l y t i c organism which i s a l s o commonly r e s i d e n t i n the fox anal sac. In a d d i t i o n , v o l a t i l e s o f the type i d e n t i f i e d i n a n a l sac s e c r e t i o n s are commonly produced when gut microorganisms are incubate Thus, the low molecula i n c l u d e 5-aminovaleric a c i d , the v o l a t i l e f a t t y a c i d s and the amines i d e n t i f i e d i n anal sac s e c r e t i o n s (45,46,47). P u t r e f i e d animal matter has formed the b a s i s f o r coyote a t t r a c t a n t s o f p o s s i b l e value i n p e s t c o n t r o l programs. Thus, a p u t r e f i e d f i s h f o r m u l a t i o n has been used as a coyote l u r e and, more r e c e n t l y , a t t e n t i o n has been d i r e c t e d t o a fermented aqueous suspension of chicken whole-egg powder, developed i n i t i a l l y as an a t t r a c t a n t f o r f l i e s (48). The odor components of t h i s m a t e r i a l have been subjected to d e t a i l e d chemical a n a l y s i s ky B u l l a r d e t a l . (49) and are r e p o r t e d t o i n c l u d e v o l a t i l e f a t t y a c i d s (77% t o t a l ; 13 a c i d s i d e n t i f i e d ) , bases (13% t o t a l , mainly trimethylamine, 9 amines i d e n t i f i e d ) , and headspace v o l a t i l e s , i n c l u d i n g e s t e r s , aldehydes, ketones, a l c o h o l s , a l k y l aromatics, terpenes and s u l f u r compounds (10% t o t a l , 76 compounds i d e n t i f i e d ) . Based on these d a t a , a s y n t h e t i c m i x t u r e , " s y n t h e t i c fermented egg" has been formulated, composed l a r g e l y of a mixture of ten v o l a t i l e f a t t y a c i d s (81%), together w i t h a d i v e r s e range of amines and other compounds (50). T h i s mixture was found to be as a t t r a c t i v e t o coyotes as the fermented p r e p a r a t i o n i t s e l f . The v o l a t i l e f a t t y a c i d component alone was found to e x h i b i t subs t a n t i a l coyote a t t r a c t a n c y a l s o (50,51) . As a f u r t h e r example o f the a t t r a c t a n c y o f m i c r o b i a l products an unfortunate case i s quoted i n the medical l i t e r a t u r e (52) o f a woman s u f f e r i n g from a s k i n c o n d i t i o n which, as the r e s u l t o f secondary i n f e c t i o n , emitted obnoxious odors which caused her t o be molested by dogs. In a d i f f e r e n t c o n t e x t , Amoore and F o r r e s t e r (5_3) quoted Linnaeus' o b s e r v a t i o n t h a t the domestic dog i s a t t r a c t e d by the odor of the p l a n t Chenopodium v u l v a r i a , the leaves o f which are r i c h (400 ppm) i n trimethy1amine. Mammalian and Environmental Fermentative Scent Sources Compared Even on the b a s i s o f present l i m i t e d knowledge, chemical and


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m i c r o b i o l o g i c a l s i m i l a r i t i e s a r e emerging f o r the r e d fox between a mammalian scent source, the anal sac, and an important e n v i r o n mental scent source, c a r r i o n . T h i s i s not t o say t h a t these scents are not d i s t i n g u i s h a b l e t o the f o x . Fundamental d i f f e r ences remain t o the extent t h a t the anal sac fermentation i s c o n t r o l l e d and l i m i t e d by the l i v i n g mammal. The o b s e r v a t i o n o f s t r u c t u r e i n the a n a l sac micro-ecosystem seems t o suggest the o p e r a t i o n o f s e l e c t i v e pressures and so t o imply the e x i s t e n c e o f s p e c i f i c b i o l o g i c a l f u n c t i o n s o f the a n a l sac o f adaptive value f o r the fox. Superimposed on the general a t t r a c t a n c y which fermentative scent sources e x e r t f o r c a n i d s , the mammal can convey more s p e c i f i c i n f o r m a t i o n t o the degree t h a t the a n a l sac scent source responds t o the p h y s i o l o g i c a l and s o c i a l circumstances o f the mammal. Although very l i t t l e research has y e t been conducted on any such scent source i such processes i n othe through hormonal r e g u l a t i o n o f s u b s t r a t e a v a i l a b i l i t y , the rhesus monkey i s able t o r e g u l a t e the p r o d u c t i o n o f v o l a t i l e compounds by i t s v a g i n a l m i c r o f l o r a so t h a t m i c r o b i a l scent p r o d u c t i o n from t h a t source r e f l e c t s the r e p r o d u c t i v e s t a t e o f the monkey (16). And, i n the r a t , maternal pheromone, the scent which a t t r a c t s the pups t o the mother, a r i s e s from the a c t i o n o f b a c t e r i a on food i n the cecum. This i s r e g u l a t e d by high p r o l a c t i n l e v e l s which s t i m u l a t e the l a c t a t i n g r a t t o high food and water consumption and l e a d t o the p r o d u c t i o n and emission o f excess cecotrophe, thus p r o v i d i n g the chemical s i g n a l (54) . The p o s s i b i l i t y o f mammalian fermentative scent sources a c q u i r i n g i n d i v i d u a l o r group r e c o g n i t i o n value has a l s o been d i s c u s s e d i n r e l a t i o n t o the r e d fox and the I n d i a n mongoose, although the experimental evidence a t present a v a i l a b l e i n support of t h i s o c c u r r i n g i n these species i s n o t strong (5,35,55). The s i g n a l l i n g f u n c t i o n ( s ) o f a n a l sac s e c r e t i o n i n the l i f e of the f o x i n i t s n a t u r a l environment remain(s) u n c l e a r , r e f l e c t ing the p a u c i t y o f i n f o r m a t i o n which e x i s t s on the chemical ecology o f w i l d mammalian s p e c i e s . As w i t h u r i n e , i t probably conveys a v a r i e t y o f messages i n a v a r i e t y o f circumstances (56). P r e l i m i n a r y f i e l d observations (_4) i n d i c a t e t h a t the s e c r e t i o n may be deposited w i t h feces o r t h a t i t may be emitted without d e f e c a t i o n when the f o x i s alarmed. Anal sac evacuation has been observed a t t e r r i t o r i a l boundaries f o l l o w i n g e v i c t i o n o f an i n t r u d e r . However, an exhaustive a n a l y s i s o f the s i g n a l l i n g r o l e of t h i s s e c r e t i o n has y e t t o be undertaken. Our l a b o r a t o r y s t u d i e s on the b e h a v i o r a l response o f the r e d f o x t o a n a l sac s e c r e t i o n have, l i k e those o f Doty and Dunbar w i t h beagle a n a l sac s e c r e t i o n (57), proved o f l i m i t e d v a l u e . T h i s i s not unexpected when the gross nature o f the responses assessed (frequency and d u r a t i o n o f i n v e s t i g a t i o n ) i n a r t i f i c i a l surroundings are compared w i t h the complexity o f the r e l a t i o n s h i p o f a w i l d mammal to i t s n a t u r a l environment. I d e a l l y , one would i n v e s t i g a t e the


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d e t a i l e d e f f e c t o f chemical s i g n a l s on the e n t i r e b e h a v i o r a l r e p e r t o i r e o f w i l d animals. F i e l d s t u d i e s on the s i g n i f i c a n c e o f scent marks i n the l i f e o f w i l d mammals remain few. Notable examples are those o f Peters and Mech on the wolf (58) and of Henry on the r e d f o x (56). Behavioral

studies

Basing our methods on those employed by L i n h a r t and Knowlton (59) using fermented egg product as a coyote a t t r a c t a n t , we have s t u d i e d the response o f the f o x t o fermented products r e l a t e d i n some measure t o anal sac s e c r e t i o n . A d e t a i l e d account o f t h i s work w i l l be p u b l i s h e d s h o r t l y (Macdonald, Hough, B l i z a r d and Perry, i n preparation). Fermented egg product i s produced by exposing an aqueous suspension o f chicken whole-eg temperature f o r 7 t o 1 micro-organisms from the a i r , and these b r i n g about fermentation. As w e l l as being u n c o n t r o l l e d , t h i s method o f i n o c u l a t i o n excludes s t r i c t anaerobes which are among the most e f f e c t i v e odor producers. I n our i n i t i a l experiments, we have compared the a t t r a c t a n c y o f a f o x t i s s u e e x t r a c t (FE), incubated a n a e r o b i c a l l y w i t h an inoculum o f f r e s h , a n a e r o b i c a l l y c o l l e c t e d a n a l sac s e c r e t i o n ( F E I ) , w i t h (a) a f o x t i s s u e e x t r a c t c o n t r o l , not so i n o c u l a t e d and incubated (FEC); (b) r e d f o x ($£) u r i n e ; (c) d i s t i l l e d water. FEI possesses the advantages o f being r e a d i l y prepared i n bulk f o r f i e l d t e s t i n g w h i l e m i r r o r i n g , probably more c l o s e l y than other r e a d i l y a v a i l a b l e fermentations, features o f f o x a n a l sac s e c r e t i o n s . The f o x t i s s u e e x t r a c t medium was prepared by simmering f r e s h r e d fox meat (1.2 kg h i n d l e g m u s c l e / l i v e r ) w i t h water f o r one hour, d r a i n i n g the e x t r a c t , a d j u s t i n g i t s pH t o 8.2 and f i l t e r i n g . To 1000 ml o f the r e s u l t i n g l i q u i d , 20 ml c y s t e i n e / s u l f i d e s o l u t i o n (1.5 g L - c y s t e i n e hydrochloride/1.5 g Na^S^HÔ i n 100 ml aqueous s o l u t i o n ) were added f o l l o w e d by 1 g L-methionine, 5 g NaCl and 10 g D i f c o Bacto-peptone t o y i e l d the f i n a l f o x t i s s u e e x t r a c t medium. Incubations were conducted a t 37 f o r 60 hours. Anaerobic i n c u b a t i o n s s i m i l a r l y prepared from other media using a n a l sac i n o c u l a , were a l s o t e s t e d . These media were Robertson's meat broth (RM), c a s e i n a c i d hydrolysate medium (CHY) and an egg y o l k medium (EY) ( f r e s h egg y o l k / g l u c o s e / D i f c o b r a i n heart i n f u s i o n b r o t h ; 5/2/100 w/w/w). A f t e r i n c u b a t i o n , a l l fermentations were stopped by the a d d i t i o n o f excess s o l i d sodium c h l o r i d e , and maintained a t 4°C u n t i l r e q u i r e d f o r t e s t i n g . Tests were conducted i n a small deciduous woodland where the movements o f many o f the r e s i d e n t foxes had e a r l i e r been establ i s h e d by r a d i o - t r a c k i n g (_4) . Probably a t l e a s t e i g h t a d u l t foxes (2 male, 6 female) were using the experimental area n i g h t l y . From d i r e c t observations and f i e l d s i g n s , the most h e a v i l y used t


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fox paths through the woodland were i d e n t i f i e d and these were used i n the subsequent t r i a l s . V e g e t a t i o n was c l e a r e d from two c i r c u l a r areas (60 cm diameter) on opposite s i d e s o f , and a d j o i n i n g , the s e l e c t e d fox path. T h i s process was repeated a t i n t e r v a l s o f 5 m u n t i l s i x p a i r s o f such c i r c l e s were prepared. I n each case, l e a f l i t t e r was removed and the top s o i l f i n e l y s i f t e d u n t i l i t formed a s u f f i c i e n t l y deep l a y e r t o r e l i a b l y d e t e c t i n d i v i d u a l f o o t p r i n t s . In a d d i t i o n , f u r t h e r s i f t e d areas were prepared spanning the path between each c i r c l e p a i r . In the l a t e afternoon o f the f i r s t experimental day ( A p r i l ) , a s m a l l cane b e a r i n g a c i r c u l a r f i l t e r paper (4.25 cm) was p o s i t i o n e d i n the c e n t e r o f each earthen c i r c l e , standing a t a height o f some 15 cm and an a l i q u o t (1 ml) o f a g i v e n odorant was dropped on t o the f i l t e r paper. On each subsequent day a t a s i m i l a r time the earthe the canes were r e p l a c e paper and a new odorant, and the s o i l c a r e f u l l y s i f t e d a g a i n . The d i s t r i b u t i o n o f odorant along the path ( s p a c i a l d e n s i t y and l o c a t i o n on v i s u a l l y conspicuous objects)resembled the d i s t r i b u t i o n o f scent (urine) marks commonly produced by a fox i n t h i s environment. Each experiment compared f o u r odorants i n each o f the s i x p o s s i b l e b i n a r y combinations, one combination f o r each c i r c l e p a i r . Odorants were thus t e s t e d i n p a i r s , randomizing t h e sequence o f p r e s e n t a t i o n o f each p a i r along the path from n i g h t to n i g h t . The experiment was continued u n t i l every p a i r e d combination o f odors had been exposed t o foxes f o r 10 n i g h t s , d i s c o u n t i n g n i g h t s when an absence o f t r a c k s on the s i f t e d s e c t i o n s between the c i r c l e s r e v e a l e d t h a t no foxes had f o l l o w e d t h a t path t h a t n i g h t . Thus, the s i x p o s s i b l e p a i r s o f f o u r odorants were compared 10 times. A d i s c u s s i o n o f the s t a t i s t i c a l a n a l y s i s o f p a i r e d comparisons o f t h i s type has been g i v e n by Brown (60). In the f i r s t experiment, subsequently repeated i n t r i p l i c a t e w i t h s i m i l a r r e s u l t s , fox t i s s u e e x t r a c t i n c u b a t i o n (FEI), f o x t i s s u e e x t r a c t c o n t r o l (FEC), fox u r i n e and water were compared. FEI was found t o e l i c i t a s i g n i f i c a n t l y g r e a t e r response than e i t h e r fox u r i n e o r the two c o n t r o l s . T h i s i s o f c o n s i d e r a b l e i n t e r e s t as q u a l i t a t i v e o b s e r v a t i o n s on fox behavior have a l r e a d y i n d i c a t e d the g r e a t a t t r a c t a n c y o f a l i e n fox u r i n e ( 4 ) . T h i s r e s u l t was i n d i c a t e d i n two ways. F i r s t , a simple nominal ranking based on the number o f n i g h t s odorants were v i s i t e d over the e n t i r e experimental sequence i n d i c a t e d t h a t FEI was v i s i t e d on s i g n i f i c a n t l y more n i g h t s than FEC o r water, and t h a t u r i n e was v i s i t e d on s i g n i f i c a n t l y more n i g h t s than water. Thus, i n a t y p i c a l experimental sequence, FEI was v i s i t e d on 21 n i g h t s ; u r i n e , 16; FEC, 11; y p t e r , 7 (maximum s c o r e , 30). These d i f f e r e n c e s were not random (}( , 8.05; d f , 3; p< 0.05) . In order t o rank the a t t r a c t a n c y on an o r d i n a l s c a l e , a s c a l i n g f a c t o r based on the f o o t p r i n t count w i t h i n 30 cm o f an odorant was employed. S c a l e d mean scores were obtained f o r each


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odorant on each n i g h t using a m a t r i x o f d i f f e r e n c e scores f o r each odor (60). O v e r a l l s c a l e d mean scores f o r each odorant (the means o f the n i g h t l y s c a l e d mean s c o r e s , taken over the t e n nights) a r e given i n Table I I I . Randomized b l o c k a n a l y s i s o f v a r i a n c e showed t h a t i n s p i t e o f v a r i a t i o n s between i n d i v i d u a l n i g h t s , d i f f e r e n c e s between scores were s t a t i s t i c a l l y s i g n i f i c a n t . Table I I I . A t t r a c t a n c y t o odorants Experiment

O v e r a l l s c a l e d mean scores f o r odorant FEI

Red f o x ; f o o t p r i n t number 1.55 F e r r e t ; frequency 0.53 Ferret; t o t a l d u r a t i o n (sec) 3.45 F

E

(a) [ c x ]

(see t e x t )

3 / 2

7

=

3

'

5 1

(

P

=

urine

FEC

water

0.38

-0.6

-1.3

-9.15

-9.05

14.75 °-

Randomized b l o c k a n a l y s i s o f variance a error MS F 15.41 1316.97

1.73

8.91

38.23

34.45

0 1 )

The a n a l y s i s o f t h e s i g n i f i c a n c e o f d i f f e r e n c e s between p a i r s of s c a l e d mean scores p r e s e n t s s e r i o u s s t a t i s t i c a l problems which have been overcome using Brown's method (60). F o r a f u l l d i s c u s s i o n , see Macdonald, Hough, B l i z a r d and P e r r y , i n p r e p a r a t i o n . The r e s u l t s a l l o w the f o l l o w i n g preference rank t o be concluded FEI^

f o x u r i n e > FEC s

water

The measure o f a t t r a c t a n c y used was very crude and, i n order to begin t o c l a r i f y the s p e c i e s s i g n i f i c a n c e o f these r e s u l t s , we examined the responsesof a f e r r e t , Mustela f u r o , t o these substances. Odorants were presented t o a male f e r r e t i n p a i r s (randomized p o s i t i o n and order d u r i n g 60 (10 minute) t r i a l s . A l i q u o t s (1 ml) o f each odorant were presented on two b l o c k s covered w i t h f i l t e r paper. T r i a l s were continued u n t i l t e n r e p l i c a t e s o f each o f the s i x b i n a r y combinations o f the f o u r odorants had been performed. Duration and frequency o f odorant i n v e s t i g a t i o n were noted. The f e r r e t showed c o n s i d e r a b l e i n t e r e s t i n the b l o c k s bearing FEI and f o x u r i n e , r e l a t i v e t o those b e a r i n g FEC and water, repeatedly v i s i t i n g these b l o c k s d u r i n g each t r i a l and s l i t h e r i n g across them, sometimes u r i n a t i n g and probably a l s o l e a v i n g g l a n d u l a r s e c r e t i o n s . The o v e r a l l s c a l e d mean scores o f both the v a r i a b l e s measured are g i v e n i n Table I I I . The r e s u l t s o f analyses of v a r i a n c e o f both these measures i n d i c a t e s h i g h l y s i g n i f i c a n t d i f f e r e n c e s between these scores (p < 0.001). Thus, as w i t h the f i e l d t r i a l s w i t h the f o x , f o x u r i n e and FEI were p r e f e r r e d t o FEC and water, but the ranking was d i f f e r e n t .


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An a n a l y s i s as p r e v i o u s l y gives the f o l l o w i n g preference based on frequency o f i n v e s t i g a t i o n .

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rank

Fox u r i n e > FEI > FEC = water Although these f i n d i n g s are p r e l i m i n a r y and f u r t h e r s t u d i e s are c u r r e n t l y i n progress, they do i n d i c a t e t h a t FEI i s very a t t r a c t i v e t o foxes (although they do not i n d i c a t e the f u n c t i o n a l s i g n i f i c a n c e o f t h i s a t t r a c t i o n ) and t h a t t h i s a t t r a c t i o n a r i s e s as the r e s u l t o f anaerobic i n c u b a t i o n . The d i f f e r e n c e i n ranking noted i n the experiment w i t h the f e r r e t , i f confirmed by more extensive t e s t i n g , would i n d i c a t e t h a t the a t t r a c t a n c y has a degree o f species s p e c i f i c i t y and t h i s , i n t u r n , would open the way t o f u r t h e r i n v e s t i g a t i o n s o f both academic and a p p l i e d importance on the b i o l o g i c a l f u n c t i o n s o f scent s i g n a l s . I n t h i s context, i t i s o f i n t e r e s s t u d i e s using anaerobi the other media (RM, CHY, EY) have revealed only weak f o x a t t r a c t a n c y compared w i t h the fox t i s s u e e x t r a c t i n c u b a t i o n . I s t h i s because the fox t i s s u e e x t r a c t i n c u b a t i o n possesses some f e a t u r e i n common w i t h i n c u b a t i o n s o c c u r r i n g i n the f o x e s own anal sac? These phenomena r e q u i r e f u r t h e r c o n f i r m a t i o n and c l a r i f i c a t i o n . However, l a b o r a t o r y t e s t s so f a r undertaken w i t h four a d u l t male foxes using an olfactometer t o measure frequency and d u r a t i o n o f v i s i t s t o odor p o r t s a r e broadly i n accord w i t h these f i e l d t r i a l s (Macdonald, Hough, B l i z a r d and P e r r y , i n preparation). 1

Literature Cited 1. Pederson,C.S., "Microbiology of Food Fermentations," AVI Publishing Company, Inc., Westport, Conn., 1971. 2. Margalith,P. and Schwartz,Y., ADV. APPL. MICROBIOL. (1970) 12, 35-88. 3. Webb,A.D. and Muller,C.J., ADV. APPL. MICROBIOL. (1972) 15, 75-146. 4. Macdonald,D.W., D.Phil. Thesis, Oxford University, U.K. 1977. 5. Albone,E.S., Gosden,P.E. and Ware,G.C. "Chemical Signals in Vertebrates," eds. D.Müller-Schwarze and M.M.Mozell, pp.35-43, Plenum, New York, 1977. 6. Mykytowycz,R., "Communication by Chemical Signals," eds. J.W.Johnston, D.G.Moulton and A.Turk, pp.327-360, AppletonCentury-Crofts, New York, 1970. 7. Johnston,R.Ε. BEHAV. BIOL. (1974) 12, 111-117. 8. Müller-Schwarze,D. ANIM. BEHAV. (1971) 19, 141-152. 9. Berüter,J., Beauchamp,G.K. and Muetterties,E.L, BIOCHEM. BIOPHYS. RES. COMMUN. (1973) 53, 264-271. 10. Vandenbergh,J.G., Finlayson,J.S., Dobrogosz,W.J., Dills,S.S. and Kost,T.A. BIOL. REPROD. (1976) 15, 260-265.


90


11. Müller-Schwarze,D. and Mozell,M.M. (eds.) "Chemical Signals in Vertebrates," Plenum, New York, 1977. 12. Albone,E.S. CHEMISTRY IN BRITAIN (1977) 13, 92-96, 99, 112. 13. Baer,G.M., "The Natural History of Rabies," vol.2, ed. G.M.Baer, pp.261-266, Academic Press, New York, 1975. 14. Ewer,R.F., "The Carnivores," Weidenfeld and Nicholson, London, 1973. 15. Schaffer,J., "Die Hautdrüsenorgane der Säugetiere," Urban und Schwarzenberg, Berlin and Vienna, 1940. 16. Michael,R.P., Bonsall,R.W. and Zumpe,D. VITAMINS AND HORMONES, (1976) 34, 137-186. 17. Singer,A.G.,Agosta,W.C.,O'Connell,R.J., Pfaffmann,C., Bowen,D.V. and Field,F.H. SCIENCE (1976) 191, 948-950. 18. Miller,Α., Scanlan,R.A., Lee,J.S. and Libbey,L.M. APPL. MICROBIOL. (1973) 26, 18-21. 19. Rasmussen,R.A. TELLU 20. Hungate,R.E., "The Rume New York and London, 1966. 21. Thiessen,D.D., Regnier,F.E., Rice,M., Goodwin,M., Isaacks,N., and Lawson,N. SCIENCE (1974) 184, 83-85 22. Müller-Schwarze,D., Müller-Schwarze,C., Singer,A.G. and Silverstein,R.M. SCIENCE (1974) 183, 860-862. 23. Montagna,W. and Parks,H.F. ΑΝΑΤ. REC. (1948) 100, 297-315. 24. Spannhof,I. FORMA ET FUNCTIO (1969) 1, 26-45. 25. Albone,E.S. and Grönneberg,T.O. J. LIPID RES. (1977) 18, 474-479. 26. Greer,M.B. and Calhoun,M.L. AMER. J. VET. RES. (1966) 27, 773-781. 27. Andersen,K.K. and Bernstein,D.T. J. CHEM. ECOL. (1975) 1, 493-499. 28. Schildknecht,H., Wilz,I., Enzmann,F., Grund,N. and Ziegler,M. ANGEW. CHEM. INT. ED. ENGL. (1976) 15, 242-243. 29. Albone,E.S., Eglinton,G., Walker,J.M. and Ware,G.C. LIFE SCI. (1974) 14, 387-400. 30. Gosden,P.E. and Ware,G.C. J. APPL. BACT. (1976) 41, 271-275. 31. Gosden,P.E., Ph.D. Thesis. Bristol University, U.K. 1977. 32. Gosden,P.E. and Ware, G.C., J. APPL. BACT. (1977) 42, 77-79. 33. Sneath,P.H.A. and Soldai,R.R., "Numerical Taxonomy," W.H.Freeman & Co., San Francisco, 1973. 34. Celesk,R.A., Asano,T. and Wagner,M. PROC. SOC. EXP. BIOL. MED. (1976) 151, 260-263. 35. Albone,E.S. and Perry,G.C. J. CHEM. ECOL. (1976) 2, 101-111. 36. Preti,G., Muetterties,E.L., Furman,J.M., Kennelly,J.J. and Johns,B.E. J. CHEM. ECOL. (1976) 2, 177-186. 37. Sokolov,V.E., Chikil'din,B.S. and Zinkevich,E.P. DOKL. AKAD. NAUK. SSSR. (1975) 220, 220-222. 38. Gorman,M.L., Nedwell,D.B. and Smith,R.M. J. ZOOL. (1974) 172, 389-399. 39. Beruter,J., Beauchamp,G.K. and Muetterties,E.L. PHYSIOL. ZOOL. (1974) 47, 130-136.


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40. Michael,R.P. and Bonsall,R.W., "Chemical Signals in Vertebrates," eds. D.MUller-Schwarze and M.M.Mozell, pp. 251-271, Plenum, New York, 1977. 41. Sokolov,V.E. and Khorlina,I.M. DOKL. AKAD. NAUK. SSSR. (1976) 228, 225-227. 42. Albone,E.S. and Fox,M.W. NATURE (1971) 233, 569-570. 43. Albone,E.S., Robins,S.P. and Patel,D. COMP. BIOCHEM. PHYSIOL. (1976) 55B, 483-486. 44. Corry,J.E.L. J. APPL. BACT. in press 45. Brooks,J.B. and Moore,W.E.C. CAN. J. MICROBIOL. (1969) 15, 1433-1447. 46. Moss,C.W., Howell,R.T., Farshy,D.C., Dowell,V.R. and Brooks,J.B. CAN. J. MICROBIOL. (1970) 16, 421-425. 47. Mead,G.C. J. GEN. MICROBIOL. (1971) 67, 47-56. 48. Hwang,Y-S., Mulla,M.S. and Axelrod,H. J. AGRIC. FOOD CHEM. (1976) 24, 164-169 49. Bullard,R.W., Leiker,T.J. J. AGRIC. FOOD CHEM. in press. 50. Bullard,R.W., Shumake,S.A., Campbell,D.L. and Turkowski,F.J. J. AGRIC. FOOD CHEM. in press. 51. Shumake,S.A., "Chemical Signals in Vertebrates," eds. D.Müller-Schwarze and M.M.Mozell, pp.357-376, Plenum, New York 1977. 52. Liddell,K. POSTGRAD. MED. J. (1976) 52, 136-138. 53. Amoore,J.E. and Forrester,L.J. J. CHEM. ECOL. (1976) 2, 49-56. 54. Leon,M, PHYSIOL. BEHAV. (1974) 13, 441-453. 55. Gorman,M.L. ANIM. BEHAV. (1976) 24, 141-145. 56. Henry,J.D. BEHAVIOUR (1977) 61, 82-106. 57. Doty,R.L. and Dunbar,I. PHYSIOL. BEHAV. (1974) 12, 825-833. 58. Peters,R.P. and Mech,L.D. AMER. SCIENTIST (1975) 63, 628-637. 59. Linhart,S.B. and Knowlton,F.F. WILDL. SOC. BULL. (1975) 3, 119-124. 60. Brown,R.Ε. J. COMP. PSYCH. PHYSIOL, in press 61. Macdonald,D.W. pp.90-116 and Lloyd,H.G. pp.117-133 in "Rabies; the facts." ed.C.Kaplan, Oxford University Press/Corgi Books, U.K. 1977. We thank the Nuffield Foundation (ESA), the Science Research Council (Grant B/RG/6973/6; GCW/PEG), the Medical Research Council (NGH: postgraduate studentship) and the University of Bristol for financial support, and G.C.Perry, R.Blizard and R.E.Brown for valuable discussions. RECEIVED October 25, 1977.


6 Biochemical and Physiological Aspects of Animal Behavior: Taste and Smell G.V.ODELL,J.E.HALL, andW.E.McMURPHY Departments of Biochemistry and Agronomy, Oklahoma Agricultural Experiment Station, Oklahoma State University, Stillwater, OK 74074 J.E.McCROSKEY Department of Animal Sciences, University of Idaho, Moscow,ID83843 Considerable data ha to s p e c i f i c chemicals. The i n t r o d u c t i o n of certain chemicals i n t o the animals area will produce a p r e d i c t a b l e behavior response such as r e p r o d u c t i o n , f e e d i n g , alarm, attraction, r e p e l l e n c e or defense. Taste and s m e l l by animals will be the two p h y s i o l o g i c a l d e t e c t i o n routes considered in this paper. An e x c e l l e n t review on sensory r e c e p t i o n covers the literature up to the e a r l y seventies as Vol. 17 o f the s e r i e s M o l e c u l a r Biology Biochemistry B i o p h y s i c s ( 1 ) . The e d i t o r V i n n i k o v and c o - e d i t o r s Kleinzeller, Springer and Wittman has covered the reported research on c y t o l o g y , molecular mechanisms and e v o l u t i o n of vision, t a s t e , s m e l l , hearing and g r a v i t y r e c e p t i o n . Many classical molecular b i o l o g y , b i o c h e m i s t r y and b i o p h y s i c a l experiments a r e reviewed (1) (Table I). As s t a t e d earlier, t a s t e , chemicals that initiate a gustatory response, and s m e l l , chemicals that f u n c t i o n as odorant molecules have been e x t e n s i v e l y s t u d i e d , first w i t h arthropods and more r e c e n t l y w i t h v e r t e b r a t e s . Since the development o f very efficient s e p a r a t i o n techniques and h i g h s e n s i t i v e d e t e c t i o n systems, we a r e no longer l i m i t e d to t a s t e panels and t h r e s h o l d odor d e t e c t i o n by humans. Table I shows some examples of the behavior response as shown by certain species f o r a specific chemical. This very l i m i t e d l i s t does not i n c l u d e m i g r a t i o n s , rhythms and many primary s e c r e t o r y processes i n i t i a t e d by a p h y s i c a l or chemical s t i m u l u s . C e r t a i n m i g r a t i o n s are i n i t i a t e d by chemical stimuli. Rhythms of organisms may be i n i t i a t e d by p h y s i c a l changes but secondary chemical processes are d e f i n i t e l y i n v o l v e d . Glandular s e c r e t i o n s such as s a l i v a t i o n and many others r e s u l t from chemical odorant molecules o r gustatory s t i m u l i . Taste V i n n i k o v (1) c r e d i t s Lomonosov (2) w i t h d i s t i n g u i s h i n g seven d i f f e r e n t t a s t e sensations — "1. a c i d , as i n v i n e g a r ; 2. c a u s t i c , as i n g r a i n a l c o h o l ; 3. sweet, as i n honey; 4. b i t t e r , as i n t a r ; ©

0-8412-0404-7/78/47-067-092$05.00/0


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Table I Species Response t o Gustatory and O l f a c t o r y Chemical S t i m u l i Response or Condition

Species

Chemicals or

Feeding

Deer, Antelope, C a t t l e Herbivores

Essential o i l s Tannins

Reproduction

Deer Rodents

Muskone C e r t a i n grass odors (food supply)

Alarm

Deer, Antelope

Predator body odor (low molecular weight organic a c i d s )

T e r r i t o r i a l markers

Deer, Coyote, Wolf

Gland s e c r e t i o n s

Defense

Skunk, C i v e t c a t

Gland S e c r e t i o n s (skunk o i l )

Insects

Plant v o l a t i l e s (essential o i l s ) Nepetalactone (catnip)

Attractants

Cats Repellants

Insects Humans

Plant v o l a t i l e s Cadaverine, putrescine, H S (amines) 2

Death

Mammals

Amines as above



94

5. s a l t y , as i n s a l t ; 6. pungent, as i n w i l d r a d i s h ; 7. sour, as i n unripe f r u i t — . With v e r t e b r a t e s a c i d , s a l t , sweet and b i t t e r can be perceived w i t h s p e c i f i c chemcials. Table I I shows the c l a s s i f i c a t i o n o f gustatory substances as made by most researchers. 1 1

Table I I Gustatory S e n s i t i v i t y o f Vertebrates and Some I n v e r t e b r a t e s Lomonosov Ref. 2

Other ^ Researchers*

Sweet

Sweet

C o o l i n g (menthol)

Sour

Sour (acid)

Salt

Salt

Bitter

B i t t e r (quinine, a l k a l o i d s )

Commonly Accepted

(sugar)

Burning

(aminoethanol)

(salt Biting

(vanillin)

Caustic (ethyl alcohol) Pungent ( w i l d r a d i s h ) ^Personal Communication ^Reference 7 The s t r u c t u r a l o r g a n i z a t i o n o f the g u s t a t o r y receptor c e l l s and t h e i r l o c a t i o n i n mammals i s w e l l described i n V i n n i k o v s review. Farbman's (3) diagram shows the stages o f development of the t a s t e bud w h i l e Murray (4) shows t a s t e buds a r e o v a l (or bulbous) i n shape and t h e i r lengths and widths a r e described. I n the I960's researchers i s o l a t e d a " s w e e t - s e n s i t i v e " p r o t e i n from t a s t e receptor c e l l s l o c a t e d i n the p o s t e r i o r p o r t i o n of p o r c i n e and bovine tongue, D a s t o l i and P r i c e ( 5 ) . The i n t e r a c t i o n of the sweet s e n s i t i v e p r o t e i n w i t h f r u c t o s e shows a s t r a i g h t l i n e assoc i a t i o n to form a complex ( 5 ) . V i n n i k o v (1) suggests more research by modern techniques i s needed on the s w e e t - s e n s i t i v e p r o t e i n as the reported molecular weight o f 152,000 (6) seems t o be w e l l e s t a b l i s h e d . I t i s t h i s author's views that t h i s may be a subunit p r o t e i n i f one considers the s t a t e o f research on the a c e t y l c h o l i n e r e c e p t o r . A f f i n i t y chromatography may be u s e f u l i n f u t u r e research on receptor p r o t e i n s even though homogeneity i s w e l l e s t a b l i s h e d f o r the s w e e t - s e n s i t i v e p r o t e i n . A b i t t e r s e n s i t i v e p r o t e i n has been i s o l a t e d and s t u d i e d by D a s t o l i e t a l . (6). This p r o t e i n was from the p o s t e r i o r t a s t e buds o f the p o r c i n e tongue and the i n t e r a c t i o n c h a r a c t e r i s t i c s w i t h four b i t t e r compounds was made. A l a r g e number of compounds i n the sweet, s a l t , sour and b i t t e r c l a s s a r e l i s t e d by V i n n i k o v (1). The e f f e c t s of the a d d i t i o n o r n a t u r a l presence of c e r t a i n components i n p l a n t s has been shown t o determine feeding s t i m u l u s . Hedin e t a l . (7) 1


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95

r e c e n t l y compiled the f e e d i n g a c t i v i t y o f f l a v o n o i d compounds (7) and many other organics f o r the b o l l w e e v i l . Formulation of a s t i m u l a n t f e e d i n g mixture was made and no s i n g l e component was e f f e c t i v e but 52 of 286 compounds bioassayed e l i c i t e d s u b s t a n t i a l f e e d i n g a c t i v i t y . Hedin et a l . have a l s o t a b u l a t e d compound, f l a v o r and odor i n t h e i r review ( 7 ) . Very r e c e n t l y work by Berger _e_t a l . (8) shows p h e n o l i c p l a n t substances, n a t u r a l l y o c c u r r i n g cinnamic a c i d and v i n y l phenols i n h i b i t r e p r o d u c t i o n i n Microtus montanus. This same group of workers (9) have t r i g g e r e d a r e p r o d u c t i v e response i n the same organism by p l a c i n g f r e s h green wheat grass i n a nonbreeding p o p u l a t i o n . This could be an o l f a c t o r y response as w e l l as g u s t a t o r y . McMurphy (10) observe d i g e s t i b l e , greater i i b l e energy and consumed i n g r e a t e r q u a n t i t i e s than o l d e r forages." This author would suggest t h i s i s t r u e f o r most h e r b i v o r e s . I n Oklahoma there i s " N a t i v e " and "Introduced" grass s p e c i e s . The chemical composition of the n a t i v e grasses i n C e n t r a l Oklahoma has been compiled by W a l l e r et a l . (11) f o r a p e r i o d from 1947 to 1962 and provides a b a s i s f o r f u r t h e r study on gustatory response. An approach u s i n g modern s e p a r a t i o n techniques on grass preference by c a t t l e i s shown i n Tables V through V I I I . Table I I I d e s c r i b e s a gas l i q u i d column, d e t e c t o r , c o n d i t i o n s and instrument used t o separate and detect the compounds of Oklahoma n a t i v e and i n t r o d u c e d grasses. The preference was determined by hand c l i p p i n g p o r t i o n s and p l a c i n g the grasses i n c a t t l e dry feed l o t s . The grasses were a t a peak l u s h p e r i o d so t a s t e and odor were i n v o l v e d . Tables IV, V, and VI show the g r a s s , e s s e n t i a l o i l y i e l d (by d i s t i l l a t i o n and e x t r a c t i o n ) preference by c a t t l e and the number of compounds observed. Taste could not be separated from odor i n t h i s l i m i t e d study but we do see a "low p r e f e r e n c e " f o r grasses h i g h i n v o l a t i l e e s s e n t i a l o i l s . One can s p e c u l a t e the nightshade p l a n t was not consumed due to t o x i c compounds present. This a l s o suggests overgrazing o f pastures w i l l g i v e the l e s s d e s i r a b l e p l a n t s an advantage. V i n n i k o v (1) concludes the t a s t e o f s a l t s depends on the c a t i o n and anion, sour o r a c i d s t i m u l u s of the hydrogen i o n , sweet and b i t t e r on i n t r a m o l e c u l a r s h i f t s of p r o t e i n r e c e p t o r s and gustatory substance complexes. He approaches the problem from a molecular b a s i s of both substance and r e c e p t o r . Exact behavior to t a s t e has covered mostly f e e d i n g and r e p r o d u c t i o n i n t h i s paper and much more f i e l d and l a b o r a t o r y research i s needed on s p e c i f i c animal s p e c i e s .



PRIMARY

PRIMARY

ODOR

TABLE

III

ODORS

AND

CHEMICAL

CAMPHORACEOUS

EXAMPLES

SUBSTANCE

EXAMPLE

MOTH

CAMPHOR

ANGELICA

PENTADECANOLACTONE

MUSKY

PHENYLETHYL

FLORAL

ETHYL

ROOT

OIL

ROSES

METHYL

CARBINOL

MINT

METHONE

PEPPERMINTY

REPELLANT

CANDY

ETHERAL

PUNGENT

FORMIC

TABLE ODOR

EGG

IV

SENSATION

PRIMARY

ET

BAD

BUTYLMERCAPTAN

PUTRID

AMOORE

VINEGAR

ACID

ODORS

AL.

VIA

HEDIN

ET

AL.

VINNIKOV

CAMPHORACEOUS

CAMPHORACEOUS

(1,8-CINEOLE)

PUNGENT

PUNGENT

(BENZYLAMINE)

ETHEREAL

AROMATIC

FLORAL

FLORAL

(STYRENEGLYCOL)

(FERULIC

ACID)

(METHONE)

MINTY

PEPPERMINTY

MUSKY

MUSKY

PUTRID

PUTRID

(H S

SWEATY

(ALPHA-KETOBUTYRIC

(CYCLOPENTADECANONE)

2

OR

NICOTINE)

ACID)


ODELL ET AL.

Taste and Smell

TABLE V

COLUMN Column 4 % Injector

DESCRIPTION

OV-17 on

GC-Q;

2 4 ' X 1/4",

Detector

temp.

260° C. ( H

Flame )

temp.

120° C

Helium

flow,

4 3 ml / min ( gauge

Instrument,

to

2

Column

Sample

Glass

temp. 180° C.

200° C.

Barber - Coleman,

at

l°C./min.

setting 7 0 )

Series

5000

siz

Τ Λ Β Ι Ι VI

OIL

YIELD AND COMPOUNDS OBSERVED WITH SELECTED GRASSES

Common Name Marestail Prairie

Threeawn

Preference

Oil Isolated mg/kg

Compounds Observed

Low

2297

55

Low

67

64

Low

2730

51

Silver Leaf Nightshade

Low

99

54

Piper

High

30

63

Mod

39

59

Low

35

55

Louisiana

Sweet T. E.

Sagewort

Sudangrass Sudangrass Haygrazer

Broomweed

Very

Low

3851

44



TABLE V I I

ESSENTIAL OIL YIELD AND COMPOUNDS OBSERVED WITH S E L E C T E D G R A S S E S

Sample Description Big

Bluestem

Switch Little

grass

Preference

Oil Isolated mg/kg

Compounds Observed

High

17

25

58

64

High

33

48

59

69

Bluestem

Weeping

lovegrass

Johnson

grass

Prairie

Threeawn

Western Caucasian

Ragweed Bluestem

Very

High

Low (Fair to) Low "Medium"

181

70

67

64

1524

56

37

60

TABLF V I I Ï

ESSENTIAL OIL YIELD AND COMPOUNDS O B S E R V E D WITH S E L E C T E D GRASSES

Sample Description

Preference

Oil Isolated mg/kg

Compounds Observed 63

S-Blend

Low

178

M-Blend

Low

277

69

LL-Blend

High

375

59

L-Blend I - Blend

High No Data

189

62

938

52


6.

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Smell Again V i n n i k o v (1) presents a review on t h i s s u b j e c t a t the molecular l e v e l . F u n c t i o n of the o l f a c t o r y organ i s suggested as a simple d i f f u s i o n of odorant molecules — "wafted around by a i r c u r r e n t s " — as they are v o l a t i l e . I n t e r a c t i o n w i t h r e c e p t o r s i n the o l f a c t o r y r e g i o n t r a n s m i t s an impulse to the c e n t r a l nervous system. One important p o i n t on s m e l l i s t h a t j u s t as w i t h t a s t e molecules of d i f f e r e n t s t r u c t u r e s , o f t e n t o t a l l y d i f f e r e n t molecules, have the same or d i f f e r e n t s m e l l . An example of rose odor i n r e f e r e n c e (1) s i t e s the work of Wright (12) where rosetone, phenylethanol, g e r a n i o l and p e l a r g o l are compounds w i t h v e r y d i f f e r e n t s t r u c t u r e s but the same s m e l l . He a l s o a t t r i b u t e s camphor s m e l l to camphor (12). Other r e s e a r c h s i m i l a r s t r u c t u r e w i t h a d i f f e r e n t o l f a c t o r y response. I n 1929 Braun (13) s t u d i e d a s e r i e s of ketones where the c a r b o n y l moved from carbon two through carbon s i x of an eleven carbon ketone. The odor ranged from rue to f r u i t y f o r d i p e n t y l ketone. These observations do not d i r e c t l y r e l a t e to behavior as t a b u l a t e d i n Table I of t h i s paper but are important on s t r u c t u r e and s m e l l . Table I I I covers a l i s t of primary odors w i t h examples (1,7). The l i s t i n g i s c u r r e n t but animal behavior i s s t i l l not covered. Amoore (14) a l s o l i s t s primary odors and chemical examples w i t h substance t a b u l a t e d i n Table IV. Amoore et a l . a r t i c l e presents the concept of o l f a c t o r y r e c e p t o r s i t e s i n t o which molecules must f i t to g i v e the odor response. A molecule could f i t one or more s i t e ( s ) to show a v a r i a b l e odor combination (14). E s s e n t i a l o i l s or v o l a t i l e o r g a n i c s of p l a n t s has a l r e a d y been shown to a f f e c t f e e d i n g , choice of food and r e p r o d u c t i o n . There i s no q u e s t i o n that organic v o l a t i l e s are r e p e l l e n t s , a t t r a c t a n t s , alarm, defense and other behavior s t i m u l i f o r animals. The word "pheremone" i n d i c a t e s a chemical t h a t e l i c i t s a s p e c i f i c animal response through o l f a c t o r y s t i m u l u s (14, 15). The f e l i n e a t t r a c t a n t , nepetalactone, has been thoroughly s t u d i e d as to s t r u c t u r e , occurrence and metabolism by W a l l e r and coworkers (16). The b i o l o g i c a l a c t i v e component i s a b i c y c l i c monoterpene l a c t o n e found i n Nepeta c a t a r i a w i t h the common name c a t n i p (17). An alarm substance has been found by the same group f o r ants (18). The i r i d o l a c t o n e s could be c l a s s e d as defense chemicals f o r t h i s s o c i e t y i n s e c t and i s s t r u c t u r a l l y r e l a t e d to c a t n i p . Another ant uses 6-methyl-5-hepten-2-one. F i e l d observers are w e l l aware of alarm substances from predators that are a i r b o r n e o r g a n i c s . Low molecular weight o r g a n i c a c i d s (sweaty), aldehydes, ketones, a l c o h o l s and amines could serve. Many a t t r a c t a n t s have been r e p o r t e d f o r i n s e c t s and would be d i f f i c u l t to d i s t i n g u i s h from t e r r i t o r i a l markers (muskone-musk


100


deer and c i v e t o n e f o r c i v e t c a t s ) . Reproduction, a t t r a c t a n t s and t e r r i t o r i a l markers become one i n these cases. I t i s a l s o o f i n t e r e s t that the p u t r i d amines of dead animals become feeding a t t r a c t a n t s f o r bear, raccoons, opossum and perhaps buzzards. C a r r i o n b e e t l e s a r e a t t r a c t e d to dead animals. Again V i n n i k o v (1) has thoroughly reviewed the p h y s i o l o g i c a l s t r u c t u r e of the o l f a c t o r y system i n mammals and arthropods. In c o n c l u s i o n we must say the o l f a c t o r y system ranks above gustatory s t i m u l i i n animal behavior. T h i s author w i l l c l o s e with a p o l o g i e s to the many r e s e a r c h e r s whose r e p o r t s were not i n c l u d e d i n t h i s b r i e f review. Other p a r t i c i p a n t s i n t h i s symposium w i l l cover much of t h i s work. Literature Cited (1)

(2) (3) (4) (5) (6) (7) (8) (9) (10)

(11)

(12) (13) (14)

(15) (16) (17)

V i n n i k o v , Ya. V., M o l e c u l a B i o p h y s i c s , V o l . 17 New York, 1974 Chapters V and V I . Lomonosov, --via r e f e r e n c e (1) 1752-1757 L e c t u r e notes. Farbman, A. I . , Developm. Biol. 11, 110 (1965a). Murray, R. G. and Murray, Α., Jour. of U l t r a s t r u c t u r e Res. 19, 337 (1967). D a s t e l i , F. R. and P r i e c , S. Science 154, 905 (1966). D a s t o l i , F. R., Lopickes, D., P r i c e , S., Biochemistry 7, 1160 (1968). Hedin, P. Α., M i l e s , L. R., Thompson, A. C. and Minyard, J . P. Jour. Ag. and Food Chem. 16, 505-513 (1968). Berger, P. J . , Sanders, Ε. Η., Gardner, P. D., and Negus, N. C., Science 195, 575-577 (1977). Negus, N. C. and Berger, P. J . , Science 196, 1230-1231 (1977). McMurphey, W. E. "The Grasses and Grasslands of Oklahoma", Annals of the Oklahoma Academy of Science, E d i t o r s J . R. Estes and R. J . Tyrl, P u b l . Robert Noble Res. Fdn., Ardmore, Okla., 1976. W a l l e r , G. R., Morrison, R. D. and Nelson, A. B. "Chemical Composition o f Native Grasses in C e n t r a l Oklahoma from 1947 to 1962", B u l l e t i n B-697, Oklahoma A g r i c u l t u r e Expt. S t a . , S t i l l w a t e r , Okla., 1976. Wright, R. Η., "The Science of Smell", A l l e n & Unwin, London 1964. Braun, J . V., Kroper, Η., Wienhaus, Η., Ber. Dtsch. Chem. Ges. 62, 2880 (1929). Amoore, J . Ε., Johnston, J . W., and Rubin, Μ., "The S t e r o chemical Theory of Odor", Scientific American, Feb. 1964, W. H. Freeman and Co., San F r a n c i s c o . Wilson, E. 0., "Pheromones", Scientific American, May 1963, W. H. Freeman and Co., San F r a n c i s c o . W a l l e r , G. R., P r i c e , G. Η., and M i t c h e l l , E. D., Science 164, 1281-1282 (1969). Regnier, F. E., Eisenbraun, Ε. J . and W a l l e r , G. R., Phyto-


6.

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(18)

Taste and Smell

101

chemistry 6, 1271 ( 1 9 6 7 ) . McGurk, D. J . , F r o s t , J . , Waller, G. R., Eisenbraun, E. J . , V i c k , Κ., Drew, W. A. and Young, J . , J . Insect P h y s i o l . 1 4 , 841-845

(1968).

RECEIVED October 25, 1977.


7 Flavor Chemistry of Carnivore Taste Systems JAMES C. BOUDREAU and THOMAS D. WHITE Sensory Sciences Center, Graduate School of Biomedical Sciences, University of Texas at Houston, TX 77030

The Carnivores

The animals c l a s s i f i e d as carnivores constitute a diverse group of mammals including such animals as weasels, foxes, hyaenas, bears, leopards and pandas. Living and extinct species of carnivores are often grouped into two superfamilies on the basis of distinctions in skulls and teeth, with one superfamily possessing a short jaw and the other a long jaw. The carnivores with the short jaw often have retractable claws which are used to grasp the prey (1), whereas the long jawed carnivores grasp prey with the teeth. Examples of species from these two carnivoral superfamilies are the cat and the dog (Figure 1). The primary distinguishing feature of all carnivores i s the possession of enlarged canines and modified cheek teeth known as carnassials (Fig. 1). The canines are used for holding or crushing, and puncturing prey; the carnassial teeth for s l i c i n g the carcass into bite sized pieces for ingestion. Although usually considered primarily in terms of their meat eating propensities, the existing species of carnivores include only a small number of species deriving sustenance t o t a l l y from the devouring of other animals. The majority of the carnivores are omnivorous in their d i e t , consuming plant as well as animal foods (2). The giant pandas, though possessing carnassial teeth, are actually herbivorous in their d i e t , and therefore are noncarnivorous carnivores. Among the most carnivorous of the land carnivores are the f e l i d s , and even they consume small quantities of plant foods, either intentionally or accidently. Grass is a common feature in f e l i d feces. Small herbivorous animals are eaten e n t i r e l y , and herbivore intestines and their contents may be relished by the felids (3). More typical of the majority of the carnivores are the caenids, for whom plant materials may form a large part of the diet. Coyotes, for instance, may exist mainly through the ingestion of plant foods at certain times of year (4). The major constituents of the natural diet of the dog and cat would be vertebrates, invertebrates and plants (Figure 2). Food ©

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Figure 1. The skulls of the cat and dog with the shearing teeth, the carnassials, indicated

INPUTS

OUTPUTS

Figure 2. Diagram of some of the major inputs and outputs of typical carnivores in a natural nutritional ecosystem


104


s e l e c t i o n i s both w i t h i n and among these major groups of foodstuffs. Food S e l e c t i o n and Chemical Senses Food s e l e c t i o n by c a r n i v o r e s i s a complicated process i n v o l v ing a complex of p r e - e s t a b l i s h e d behavior patterns together with a long t r a i n i n g p e r i o d i n the l o c a l c o n d i t i o n s of prey capture. The f e l i d s , f o r i n s t a n c e , t y p i c a l l y remain with the mother u n t i l s u b a d u l t s , when t h e i r hunting s k i l l s are e s t a b l i s h e d . In the s e l e c t i o n of a p p r o p r i a t e f o o d s t u f f s the c a r n i v o r e i s aided and d i r e c t e d by the chemical c h a r a c t e r i s t i c s of the food. V e r t e b r a t e s , i n v e r t e b r a t e s , and p l a n t s vary markedly i n t h e i r chemical composition, which i s important i n d i e t s e l e c t i o n . There e x i s t s wide v a r i a t i o n i n the t a s t f bird d bird flesh deter mined by human and anima In the o r a l and nasal c a v i t i e s of the c a r n i v o r e are a v a r i e t y of chemical sensory systems. The chemical sensory systems i n the nasal c a v i t i e s measure p r i m a r i l y v o l a t i l e compounds, whereas those i n the o r a l c a v i t i e s are responsive mainly to water s o l u b l e compounds. S t r u c t u r a l l y these sensory systems can be a n a t o m i c a l l y subdivided i n t o d i f f e r e n t sensory neural p a r t s ; such as a r e c e p t o r f o r the measurement of the chemical s t i m u l u s , a p e r i p h e r a l neural encoding and t r a n s m i s s i o n s e c t i o n , and a complex c e n t r a l nervous system f o r f u r t h e r neural p r o c e s s i n g . At l e a s t two d i f f e r e n t types of chemoresponsive sensory receptors are found i n the o r a l c a v i t i e s : f r e e nerve endings and t a s t e buds. Free nerve endings are d i s t r i b u t e d throughout the o r a l c a v i t y and are s u p p l i e d by p e r i p h e r a l sensory neurons i n the t r i g e m i n a l g a n g l i o n . Taste buds may be l o c a t e d on s p e c i a l i z e d p a p i l l a e on the tongue or s c a t t e r e d on the s o f t p a l a t e , t o n s i l s , u v u l a , e p i g l o t t i s , l a r y n x and upper p a r t of the esophagus. Taste buds are innervated by p e r i p h e r a l sensory neurons i n the g e n i c u l a t e ganglion of the f a c i a l nerve, the p e t r o s a l ganglion of the glossopharyngeal nerve and the j u g u l a r ganglion of the vagus nerve. Neurons i n these g a n g l i a send information from the t a s t e buds to the c e n t r a l nervous system (Figure 3 ) . Neurophysiology of Fungiform P a p i l l a e Taste Systems The fungiform p a p i l l a e t a s t e systems c o n s i s t of t a s t e bud receptors d i s t r i b u t e d on the fungiform p a p i l l a e which are l o c a t e d on the a n t e r i o r surface of the tongue and the p e r i p h e r a l sensory neurons i n the g e n i c u l a t e ganglion of the f a c i a l nerve. These neurons c o l l e c t sensory information r e l e v a n t to the chemical nature o f food substances i n the mouth. This chemosensory i n f o r mation i s encoded i n t o a s e r i e s of pulses and t r a n s m i t t e d to the c e n t r a l nervous system over nerve f i b e r s t h a t s t r e t c h from the tongue to the c e n t r a l nervous system (Figure 3 ) . These pulse t r a i n s can be measured e l e c t r i c a l l y w i t h microelectrodes i n the



Figure 3. Diagram of some of the sensory structures involved in oral chemoreception. Pulses being sent from the periphery to the central nervous system are recorded to determine nature of effective stimuli.

106

FLAVOR CHEMISTRY O F A N I M A L FOODS

ganglion (7) and these measures can be used to determine types of active compounds. Studies on these neural systems have determined that the carnivore performs a precise chemical analysis of certain aspects of i t s prey. The electrical pulse activity of geniculate ganglion neurons has been studied (8, 9) 1n both the cat and the dog by recording discharges when the tongue was stimulated with various chemical solutions. In both the cat and the dog these chemosensory neurons have been divided into a number of functional neural groups (£, 10) on the basis of a variety of physiological measures (Table I ) . Since these different neural groups respond to different types of chemical compounds, the types of effective compounds will be described for each neural group. The cat fungiform papillae taste system has been most intensively studied and therefore i t will be described f t r s t and then compared to that of the dog. Cat Fungiform Papillae Tast Three different chemoresponsive neural groups have been distinguished in the cat geniculate ganglion on the basis of conduction velocity measures, spontaneous activity measures and their response to chemical solutions (Figure 4 and Table I ) . The most useful chemical response measure has been that to a series of discriminatory solutions (the "test series") tested on each neural unit by application to the area of the tongue known to be innervated by the unit. On the basis of the differential response of each unit to the test series and other neurophysiological measures over 90% of the chemoresponsive tongue units can be c l a s s i f i e d into three categories, which have been labeled group I, II and III (Figure 5). These groups have proved valuable for the determination of active chemical stimuli and for the comparison of species although they do not represent total neural population v a r i a b i l i t y since unclassified units exist, as does undetermined within-group v a r i a b i l i t y . Cat geniculate ganglion group I neurons preferentially innervate receptors on fungiform papillae on the rear and sides of the tongue wilth fibers of large diameter. They display low spontaneous a c t i v i t y rates and are responsive to d i s t i l l e d water and inorganic acids. In a study testing a variety of organic compounds, i t was found that group I units were discharged by compounds with undissociated carboxylic and phosphoric acid groups Q j J . A few nitrogen compounds were also effective. In a l l cases the response was pH dependent 1n that the maximum d i s charge rate was e l i c i t e d when the solution pH was at or below the pKa of the active chemical group. The compounds most active in the pH region 5.0 to 7.0 proved to be compounds with a heterocyclic nitrogen ring component (e.g. imidazole ring). Only the proton donor form of the molecule appeared to stimulate. Group II units preferentially Innervate fungiform chemoreceptors on the front and sides of the tongue with medium size


BOUDREAU AND WHITE

Carnivore Taste Systems

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Figure 4. Examples of projection zones and discharge patterns of cat geniculate ganglion chemoresponsive units. The locations of the fungiform papillae projection zones for each neural group are indicated on the schematic tongue, where each dot represents the locus projection of a single unit. On the right are examples of spontaneous and evoked discharge patterns of group I, II, and III units. S indicates approximate time of stimulus application.


108


Table I:

CAT:

Chemosensory Neural Groups Distinguished in the Geniculate Ganglion of the Dog and Cat.

Units distinguished on the basis of response to chemicals, electrical stimulation and spontaneous activity measures.

Group I Units:

Low spontaneous a c t i v i t y , short latency to electrical stimulation. Respond to malic acid, ATP, ITP, etc.

Group II Units:

High spontaneous activity rates, medium latency to electrical stimulation. Respond to L^-prol1ne, L-cyste1ne and d i - and triphos phat and inorganic salts.

Group III Units:

Low spontaneous activity rates, long latencies to electrical stimulation. Sub divided Into two groups on basis of chemical response. Subgroup IIIA respond to nucleo tides; IIIB to various oxygen compounds.

DOG:

Units distinguished by factor analysis of responses to chemical stimulation.

Group A Units: Group Β Units: Group C Units: Group D Units:

Respond to Lrprollne, L-cysteine, ITP, ATP, IDP, and NaCl Respond to L-malic acid, quinine hydrochloride, HC1 and ATP. Respond primarily to nucleotides. Respond to butyryl choline chloride, phytic a d d and quinine hydrochloride.


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BOUDREAU AND WHITE

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Figure 5. Responses of cat geniculate ganglion chemoresponsive neurons to the test series chemical solutions (30mM in distilled water). Values represent the number of spikes during the 10-sec stimulus period minus the spikes in the preceeding 10-sec period.


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fibers. Spontaneous activity rates are often high. A survey (12) of a variety of chemical substances disclosed that the most effective excitatory stimuli were certain amino acids (L-proline, L~cyste1ne, L-lysine, L-histid1ne, e t c . ) , d i - and triphosphate nucleotides and certain other substances (usually containing nitrogen). In testing compounds related to proline and histidlne i t was discovered that the heterocyclic ring components pyrro lidine and imidazole were as stimulatory as the parent amino acids. To further specify the properties of excitatory stimuli many simple heterocyclic stimuli were tested. The most stimula tory proved to be four to six member ring nonaromatic (except for imidazole) nitrogen compounds. Inhibition, or a decrease in spontaneous a c t i v i t y was also common with group II units. Common inhibitors are L-tryptophan, L-1soleucine, pyrrole and azacyclooctane. Group III units innervate chemoreceptors on fungiform papillae located primaril Latency measures to electrica f i e l d s are greater than 14 msec, indicating that the fibers are of extremely small diameter. The spontaneous activity from these neurons i s of low rate and often extremely bursty. Discharge to chemical stimulation of the tongue may start as long as one second after stimulation, and spike patterns are often quite irregular (Pigure 4). Less i s known about the chemistry of group III units than of the other two unit groups. On the basis of their response to chemical stimulation group III units have been tentatively subdivided into two subgroups: those largely res ponsive to nucleotides (IIIA) and those responsive to nucleotides and other compounds, especially butyryl choline chloride (IIIB). Units responsive to nucleotides may show responses to concentra tions less than 0.1 mM. Units responsive to butyryl choline chloride have recently been found to respond to a variety of other compounds such as v a n i l l i n , 5-hydroxypentenal, methyl mal t o i , and certain lactones. The common feature to these compounds seems to be a carbonyl group associated with a long hydrocarbon chain. The nature of group IIIB stimuli is currently being investigated. Comparison of Cat and Dog Fungiform Papillae Taste Systems The chemoresponsive neurons of the dog geniculate ganglion have been divided into four separate functional classes on the basis of factor analysis of their responses to the test series and to other chemical stimuli (9). Two of these unit classes seem quite similar to two groups distinguished in the cat, with dog class A similar to cat group I I , class Β with group I. Class D units seem similar to group IIIB units, but only a few dog class D units have been Investigated. Dog unit class C seems to consist in part of units similar to cat unclassified units and in part of units similar to cat group IIIA units. Because of the


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111

paucity of Information on class C units and their general un responsiveness, they are not further discussed in this report. The responses of dog class Β units to the test series are v i r t u a l l y identical to those of cat group I units (Figure 6). Malic acid is by far the best stimulus for both species; and the responses to other test series compounds are proportionally similar. The major differences are the smaller response shown by the dog units to butyryl choline chloride and a larger response to L-cysteine. L-h1stidine 1s the test series amino acid most excitatory to both species. Other active stimuli for both species are HC1, quinine HCl, and c i t r i c a d d . Dog class A units are similar in many of their properties to cat group II untts. In a comparison of responses to test stimuli, the most active compounds 1n the cat proved to be those most excttatory in the dog, namely the amino acids L-proline and Lcysteine (Figure 7). L-tryptophan, the most effective cat group II inhibitor, was also th Significant differences Fewer compounds seem to inhibit dog class A units. Dog class A units are also much less responsive to nucleotides than are cat group II units. In addition to the test series chemicals, a variety of amino acids (Figure 7) and nitrogen heterocycles (Figure 8) were tested on the dog class A units. As with the test series many amino acids e l i c i t e d similar responses from the two species, but the response of the dog units was less than that of the cat units to amino actds with carboxyl and hydroxyl groups on the side chains. Uhtsttdtne i s also less stimulating in the dog. As with the test serfes, fewer compounds inhibited in the dog; in fact, Lphenylalanine and L-tyrosine were excitatory in the dog. On the other hand, a comparison of the responses to simple nitrogen heterocycles reveals remarkable similarities between the two species, even though the chemicals were tested in d i s t i l l e d water solution for the dog and in a neutral saline solution for the cat. A relationship between nitrogen heterocycle pKa values, ring size, and neural response 1s seen 1n both species (Figure 8). Heterocycles with pKa values less than 7.0 are inactive or i n hibitory in both species. As in the cat group II units, dog class A units were those most responsive to NaCl. Unlike cat units, however, dog class A units also discharged to sugars, with fructose and sucrose being the most excitatory sugars (Figure 9). Other dog unit groups were unresponsive to sugar solutions. The response to sugars, even in group A units, was much less than the response shown to an equivalent concentration of L-proline (Figure 9). Dog class D units are similar in part in their response properties to cat group IIIB units. The most effective test stimulus for both species 1s butyryl choline chloride (Figure 10). The other test series chemicals stimulate both species to approximately the same degree, although compounds with phosphate



112

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DOG


116

TEST SERIES

NoHjPQ,

4$»

"T" "T" 80 60 C A T - G R O U P ΙΠ Β - N O O F SPIKES

-20

Figure 10.

I—

100

Comparison of cat group IIIB and dog class D unit responses to the test series


7.

BOUDREAU AND WHITE


117

groups are less stimulatory in the dog. As with the cat, certain oxygen compounds are quite effective stimuli for dog class D units. Some of the most active stimuli for dog class D units are furaneol, ethyl mal t o i , and methyl maltoi. Furaneol, a potent dog group D stimulus has been relatively inactive in the cat. The chemical stimulus dimensions of both unit groups have been inadequately investigated, but both dog class D and cat group IIIB units are less responsive to nitrogen compounds than the other unit groups. Flavor Chemistry By flavor chemistry is meant the chemistry of the total complement of sensory responses e l i c i t e d by a food or a food component. In human sensory research, the sensory responses are psychophysical sensations, whereas in neurophysiological studies the responses are measure the geniculate ganglion then consist of a description of the neural responses to foods and to the types of compounds present in food. The neural responses of both the dog and the cat have been examined with respect to the excitability of many of the compounds found in vertebrate tissues. The cat has been tested with more compounds than the dog, but the results will also apply in large part to the dog, since the two species are so similar with respect to the fnajority of the compounds considered. Cat taste units are responsive to water extracts of various animal tissues such as chicken, beef, and pork l i v e r (8). To examine the underlying chemical substrate of this food response, many of compounds found in animal tissues were tested on the different neural groups. The results of these studies are partially summarized in Table I I , which contains the responses of the different neural groups to compounds commonly found in relatively high concentration in vertebrate muscle tissues. The monovalent inorganic salts NaCl and KC1 excite few neurons in 50 mM concentration, although group I spontaneous discharges are inhibited. In addition group II unit discharge to amino acids is potentiated by KC1 and NaCl. At higher concentrations these salts stimulate group II units. The salts CaClj and NaH2P04 have proved to be among the most excitatory inorganic compounds tested on group II units. Amino adds selectively stimulate unit groups I and I I . Only L^histidine and, to a lesser extent, taurine stimulate group I units, whereas group II units respond to a variety of amino acids. Among the most potent group II stimuli discovered are L-proline and L-cysteine. Other amino adds stimulating group II units to a lesser extent are L-lysine, L-histidine, L-alanine and Lornithine. The amino acids L-tryptophan, L-isoleucine and Lphenylalanine tend to inhibit group II unit discharge. The dipepttdes carnosine (beta-alanyl-L-histidine) and


118


Table II:

Simplified Summary of Cat Taste Neuron Responses to Compounds Commonly Found in Flesh Foods, Solu tions 50 mM, pH 6r7. Group I Units

Inorganic Salts NaCl, KC1 CaClo NaH2P0 4

Amino Acids L-Proline L-Cysteine LrLysine L-Histidine L-Tryptophan L-Taurine Peptides Anserine Camosine Nucleotides Dirand Triphosphate Monophosphate Bases Other Thiamin Compounds Pyridine Compounds Imidazole Compounds Oxygen Compounds Creatine, Creatinine Polyamines +: «:

*-

Group II Units

+

potentiate ++ +

++

+++ ++ + ++

+

+

++ ++

++

+

+++

++ + to ++ ++

Group III Units

+

+++ ++

++ ++

+++

excite inhibit


7.

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119

anserine (beta-alanyl-methyl-L-histidine), of widespread occurrence In vertebrate tissues, are often potent stimuli for group I units. Only carnoslne stimulates group II units since N-methylation of a heterocyclic ring nitrogen typically eliminates group II response. The d i - and triphosphate nucleotides are active stimuli for a l l neural groups, although at pH 7.0 the response from group I units i s minimal since the phosphate group is largely dissociated. The dt- and triphosphate nucleotide salts are among the most potent stimuli for group II units and some group III units. Only group III units are responsive to any degree to monophosphate nucleotide solutions. The heterocyclic nucleotide bases are in general inactive except for inhibition of group II units. A variety of other compounds found in vertebrate tissues also affect taste neurons. Thiamin compounds, especially thiamin pyrophosphate, are active stimuli for both group I and group II units. Compounds with a and desmosine will ofte compounds with an imidazole ring such as 1-methyl imidazole. The major factor limiting the effectiveness of many of these compounds is the presence of a dissociated carboxyl group. Various heterocyclic compounds other than those described above will also stimulate cat taste neurons. Group I units will be activated by compounds with thlazolidine, quinoline or tsoquinoline rings. Group II units would be activated by compounds with pyrrolidine, pyrroline, piperidlne and piperazine rings, The heterocyclic rings active on either group can be separated 1n part by the pKa values of the nitrogen groups. A variety of carbonyl compounds, some derived from l i p i d s , are present in animal tissues. Other oxygen compounds present in low concentration may include various oxygen heterocycles and lactones. Some of these compounds stimulate cat group IIIB units. Since the chemistry of group III units i s inadequately understood, only the vague term "oxygen compounds" i s used to describe these stimuli. Some compounds present 1n vertebrate tissues are relatively inactive on cat taste systems. Various organic adds such as l a c t i c , pyruvic and levulinic are inactive in the proton acceptor form in which they commonly occur. Various polyamines (spermine, putrescine, etc.) are of widespread occurrence but tend to Inhibit group II neurons. Sugars (glucose, fructose, etc.) are inactive In the cat. The compounds creatine and creatinine, of common occurrence in vertebrate tissues, excite no neurons and inhibit group II units. In a few cases, mixtures of different compounds were studied. The salts NaCl and KC1 have been found to potentiate the response of group II units to NaH2P04 and to L-proline and L-cysteine. This potentiation even occurs when the NaCl solution by i t s e l f i s below threshold. The response of group I neurons to many solutions 1s attenuated by adding NaCl to the solution. In



120

a similar fashion mixing L-tryptophan with L-proline will inhibit the response of a group II unit to L-proline alone. The response of dog unit groups to the compounds listed in Table II would in general be quite similar to that illustrated for the cat. The major difference would be the reduced response of dog class A and class D units to nucleotides. Dog class D untts would also probably discharge to somewhat different oxygen compounds than cat group IIIB units. Theoretical Considerations Implicit In the designation of distinct functional sensory neural subsets of chemoresponsive units 1s that each functional unit group is selectively responsive to a distinct chemical signal or signals. These chemical signals can be represented in terms of the types of compounds stimulating, common chemical factors among the stimulants aqueous solutions of thes description proceeds from the specific to the general, depending in part on the stage of the investigation and in part on the understanding of the chemical factors involved. At least seven distinct chemical input signals are suggested from neurophysiological studies on the carnivore system (Table III), Those signals can be characterized to different degrees of generality. Cat group III Input signals are described primarily in terms of the unique chemical compounds that stimulate, whereas somewhat more general statements can be u t i l i z e d in typifying group I and group II input signals. Group II excitatory and inhibitory nitrogen heterocycles, for instance, can be characterized 1n terms of pKa values and ring size (Figure 8) The depiction of group I excitants goes even further since a l l of these compounds can be characterized as Br^nsted acids, i . e . in terms of their behavior in solution. These chemical signal descriptions must in a l l cases be considered preliminary and inadequate. In the consideration of group II stimuli for instance, l i t t l e consideration is given to inorganic salts. The representation of Input signals in terms of solution chemistry 1s most desirable, since the active molecular species is known to be a supramolecular complex formed by the interaction of the solute and water (13, 14, 15). Compounds such as Inorganic s a l t s , a d d s , sugars, and amines are known to strongly interact with water molecules (16, 17, 18). The solute i s often assumed to act through a water bridge or to have i t s properties modified by the interaction of water molecules. The limiting factor in the description of the stimulus in terms of solution chemistry is the inadequate understanding of the general structural and functional properties of solutions. The most general description of a carnivore taste signal has been achieved for the cat group I (dog class B) input signal. It has been shown that a l l of the group I stimuli can be described t


7.

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121


Table III: Major Chemical Stimulus Factors of Importance In Carnivore Fungiform Papillae Taste Systems Excitatory Factors

Example of Stimulus

Carnivore Neural Group

Nitrogen Factor

Lt-proline, L-cysteine, pyrrolidine, morpho line

Cat I I , Dog A

Br^nsted Acid

malic acid, L-histidine, Cat I, Dog Β pyridine

Oxygen Factor

butyryl choline chloride,

Cat IIIB, Dog species

Nucleotide Factor

ATP, ITP, ADP, IMP, etc.

Cat IIIA

Unknown Factor

ATP, ITP

Cat I I , Dog A Different in the two species

Nitrogen Factor

L-tryptophan, pyrrole

Cat I I , Dog A

Br0nsted Base Factor (?)

IMP, tetrasodium pyrophosphate

Cat I, Dog Β

Inhibitory Factors


122

FLAVER CHEMISTRY OF ANIMAL FOODS

as Br0nsted acids ( e . g . , carboxylic acids, Dhosphoric acids, and the protonated bases pyridine and imidazole). These compounds are known to possess the characteristic of rapid proton transfer in water (19). One suggested mechanism for this phenomenon is that a solute-water c i r c u i t is formed in such a way that a proton added to one part of the c i r c u i t f a c i l i t a t e s the transfer of a proton elsewhere in the molecular c i r c u i t (Figure 11). Human Comparisons The taste signals identified for the carnivore in Table III can 1n part be related to human taste signals (Table IV). This comparison can be made largely on the basis of the similarities in chemical stimuli active in the two species. Especially valuable in cross species comparison, have been the L-amino acids which in part selectively e l i c i t different sensations just as they selectively activat Cat group I and dog clas stimuli that e l i c i t human sour sensations, such as carboxylic and phosphoric acids. Among the amino acid solutions, aspartic acid solutions and even htstidine solutions e l i c i t a human sensation with a sour component (11J. Some of the compounds e l i c i t i n g a sweet sensation in the human stimulate group II (dog class A) units and those that inhibit often taste bad or bitter (12.). In both the cat and dog the amino acids with a sweet component (20, 21_) tend to stimulate (e.g. L-prol1ne, L-lysine, L-alanine, L-glycine and, probably, ^cysteine). Other compounds which often taste sweet are NaCl and KC1, which stimulate both cat group II and dog class A units, and sugar, which stimulates dog class A units. Inhibitory com pounds in both species include the strongly bitter amino acid (--tryptophan and quinine. Cat group III stimuli Include nucleotides, phosphate com pounds, various carbonyl compounds, phenolic compounds, oxygen heterocycles and lactones. Many of these compounds are common food substances and food additives. The human taste sensations e l i c i t e d by these compounds are of an indistinct variety and may be described with the terms pleasing, sweetish, creamy, etc. (22, 23). Nucleotides e l i c i t a sensation which the Japanese term ^mami," or deliciousness (24). It is quite probable that small fiber systems similar to those seen in the cat are present in the human. A variety of compounds have been reported to be important in meat flavors (25, 33). The most prominent naturally occurring compounds are amino a d d s , nucleotides and other phosphate com pounds, and inorganic salts. Of lesser importance are various sugars and organic acids. Especially prominent are nitrogensulfur compounds such as cysteine and thiamin. Various other compounds that have been reported to be of importance in meat flavor include the peptides anserine, carnosine and glutathione.


7.

BOUDREAU AND WHITE


123

V N - H - Q\

H

O-H- Ο

Η

V-RECEPTOR O-HΟ "

Χ

7

DIRECTION O F P R O T O N TRANSFER

Figure 11. Possible molecular circuitry asso ciated with receptor activation by a cat group I (dog class B) stimufont

Table IV: Some Suggested Relationships Between Humans and Carnivores Based on Similarities Among Chemical Stimuli Carnivore Taste Signals

Human Sensations

Excitatory Chemical Factors Nitrogen Factor (II & A) Br0nsted Acid Factor (I & B) Oxygen Factor (IIIB & D) Nucleotide Factor (IIIA) Unknown Factor (II & A)

Sweet Sour (pleasant, creamy, sweetish?) (Umami?)

Inhibitory Chemical Factors Nitrogen Factor (II & A) Br0nsted Base Factor (I & B)

Bitter ?



HUMAN

DOG & CAT BEST EXCITER

SWEETEST

CLASS A / G R R I I

Str^coo"

^s^Nzoo-

HYDROXY-L-PROLINE

L-PROLINE

BEST INHIBITOR

BITTEREST

^CHÊHCOQ-

r r f

n-f

H

H

L-TRYPTOPHAN

L-TRYPTOPHAN

BEST EXCITERS

SOUREST HO

HO

H

S

N

CLASS

C-CH -C-COO~ 1 NH 2

°

rf

H

N

C-CH -CH -C-COO~ 2

1

L-GLUTAMIC A C I D

H

V

HO

C-CH -CH -C-COO~ 2

Ο

2

3

L-ASPARTIC ACID

HO

B/GRRI

NH

L-GLUTAMIC ACID

3

V

S

^C-CH -C-COO~

2

2

°

NH

3

L-ASPARTIC ACID

Figure 12. Taste responses of carnivore and man to some amino acids. Human data obtained from Ref. 20.


BOUDREAU AND WHITE

5-THIOMETHYL FURFURAL

0^ 2-METHYL

PYRAZINE


2,4,5-TRIMETHYL OXAZOLINE

O ^ C H

4-HYDROXY-5-METHYL 3(2H)FURANONE

2-ACETYL-2-THIAZOLINE

H

P N

3

C ^ D

OH

PYRROL IDON Ε CARBOXYLIC ACID

2-ETHYL-5-METHYL

r-VALEROLACTONE

PYRIDINE

Figure 13. Some heterocyclic compounds associated with meat flavor in humans

Q

0

Η THIAZOLIDINE

PYRIDINE

PYRROLE

INDOLE

υ

Η PYRROLIDINE

METHYL MALTOL

0

Η MORPHOLINE

r-OCTALACTONE

Figure 14. Some of the most effective heterocyclic stimuli in the carnivore. Pyrrole ana indole are inhibitory.


126


Off-flavor or bitterness is usually reported for compounds such as creatine, creatinine and nucleotide bases. A mixture of several different types of compounds has been reported necessary to reconstruct meat flavors. This chemical complexity suggests that different sensory systems are being activated. Many of these compounds appear to exert their effects through human geniculate ganglion taste systems; others obviously stimulate other oral chemoresponsive systems. Raw flesh is assumed by a l l but the Japanese to have l i t t l e flavor. True "meat" flavor is assumed to develop following cooking. When flesh is cooked, various chemical changes occur, many of which result in the formation of flavor products. Particularly important are flavor compounds produced through the pyrolysis of nitrogen and nitrogen sulfur compounds, largely amino acids and through their interaction with other compounds, such as sugars and carbonyls. Also prominent are various compounds derived from reactions wit compounds have been reporte of which are illustrated in Figure 13. These heterocyclic compounds are usually assumed to function as v o l a t i l e s , affecting the nasal chemoreceptor systems. The close structural similarity between these compounds and many of those active on the carnivore taste system (Figure 14) argues for their being potent taste stimuli as well. Prominent stimuli for both human primate as well as carnivore contain 5 and 6 member heterocyclic rings. The major difference between the two groups of compounds is the preponderance of aromatic compounds in the odors (Figure 13). Literature Cited 1. Gonyea, W., Ashworth, R., J . Morphol. (1975) 145: 229238. 2. Ewer, R. F., "The Carnivores", Cornell University Press, Ithaca, N.Y., 1973. 3. Schaller, G. B., "The Serengeti Lion", The University of Chicago Press, Chicago, 1972. 4. Meinzer, W.P., Eukert, D.N., Flinders, J . T . , J . Range Manag. (1975) 28: 22-27. 5. Cott, H. B., Proc. Zool. Soc., London, (1946) 116: 371524. 6. Cott, H. B., Proc. Zool. Soc., London, (1953) 123: 123141. 7. Boudreau, J . C . , Tsuchitani, C . , "Sensory Neurophysiology," Van Nostrand Reinhold Co., N.Y., 1973. 8. Boudreau, J . C . , Bradley, B. E., Bierer, P. R., Kruger, S., Tsuchitani, C . , Exp. Brain Res. (1971) 13: 461-485. 9. White, T. D., "Neurophysiological Investigation of the Geniculate Ganglion of the Dog", M.S. Thesis, Univ. of Texas at Houston, Graduate School of Biomedical S c i . , 1976


7. BOUDREAU AND WHITE 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32.


Boudreau, J. C., Alev, Ν., Brain Research (1973) 54: 157-175. Boudreau, J. C., Nelson, T. E . , Chemical Senses and Flavor (1977) 2: 353-337. Boudreau, J. C., Anderson, W., Oravec, J., Chemical Senses and Flavor (1975) 1: 495-517. Franks, F. (ed.), "Water A Comprehensive Treatise", Vol. 1- 4, Plenum Press, N.Y., 1972-1975. Franks, F., Phil. Trans. Roy. Soc., Lond. B. (1977) 278: 33-57. Pullman, Α., Pullman, Β., Quart. Rev. Biophy. (1975) 7: 505-566. Blandamer, M. J., Burgess, J., Chem. Soc. Rev. (1975) 4: 55-75. Jones, F. M. III, Arnett, Ε. Μ., Prog. Phys. Org. Chem., (1974) 11: 263-322 Suggett, Α., J. Caldin, Ε., Gold, V., "Proton-Transfer Reactions", John Wiley & Sons, N.Y., 1975. Ninomiya, T., Ikeda, S., Yamaguchi, S., Yoshikawa, T., In: "Rep. 7th Sensory Evaluation Symposium," JUSE, 109123, 1966. Kirimura, J., Shimizu, Α., Katsuya, N., J. Agr. Food Chem., (1969) 17: 689-695. Furia, T. E . , Bellanca, N., "Fernaroli's Handbook of Flavor Ingredients," Vol. 2, CRC Press, Cleveland, Ohio 1975. Arctander, S. "Perfume and Flavor Chemicals," Vol. I-II, Published by author, Penn., 1971. Yamaguchi, S. In: "Sixth International Symposium on Olfaction and Taste, Abstracts," Paris, 1977. Dwivedi, Β. Κ., Crit. Rev. Food Tech., (1975) 5: 489-538. Hashida, W., Food Trade Rev., (1974) 44: 21-32. Hashimoto, Y., In: "The Technology of Fish Utilization" (R. Kreuzer, ed), 57-61, Fishing News (Books), London, 1965. Herz, K. 0., Chang, S. S., Adv. Food Res. (1970) 18: 2- 83. Mabrouk, A. F., In: "Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors", 146-183, Am. Ch. Soc., Wash. D. C., 1976. Schutte, L . , C.R.C. Crit. Rev. Food Tech., (1974) 4: 457-505. Solms, J., In: "Gustation and Olfaction an International Symposium", 92-110, Academic Press, N.Y., 1971. Wilson, R. Α., Agr. Fd. Chem. (1975) 23: 1032-1037.


127

128


33. 34.

Wilson, R. A . , Katz, I . , Flavor Industry (1974) 5: 2-8. Boudreau, J . C . , Oravec, J., White, T. D., Madigan, C . , Chu, S. P., Arch. Otolaryngol. (1977).

Acknowledgment This research was supported in part by NSF research grants. We were assisted in this work by J . Lucke, C. Madigan, H.V. Nguyen, J . Oravec, N. Tran, and J . Watkins. RECEIVED October 25, 1977.


8 Development of Diets for Food-Producing Animals WILLIAM CHALUPA and CLIFTON A. BAILE School of Veterinary Medicine and Monell Chemical Senses Center, University of Pennsylvania, Kennett Square,PA19348

Animal foods such a the nutritional quality of plant food sources by virtue of their high quality protein, excellent Β vitamin content and iron i n highly available form (1). The current world's animal population, quantities of food produced and current and projected requirements of animal food sources are summarized i n Tables I and I I . Nutrient

Requirements

Diets for food-producing animals performing various physio logical functions such as maintenance, growth, lactation and re production are formulated to contain specified amounts of nutri ents. In the United States, the most widely used standards are those published by various committees of the National Research Council under the auspicies of the National Academy of Sciences. In Great Britain, standards of the Agricultural Research Council are employed (2). Similar bodies i n other countries provide rec ommendations on animal nutrient requirements, but the most widely used standards are those from the United States and Great Britain. It i s important to recognize that recommendations such as those of the National Research Council are specified i n terms be lieved by the committees to be minimum requirements of animals of a given species, age, weight and productive status. No provisions or safety factors are provided to compensate for deviations of specific animal populations or influences of environment, disease, parasitism or other types of stress (2). The metabolic machinery of a l l animals must be provided with water, amino acids, energy, minerals and vitamins. In ruminants, u t i l i z a b l e nutrients are provided by a combination of dietary sources plus those synthesized by rumen bacteria and protozoa and u n t i l recently l i t t l e attention has been directed towards dietary supplies of amino acids and B-complex vitamins. However, infor mation accrued during the last decade demonstrates that ruminai outflows of these nutrients i s not always sufficient for high rates of productivity ( 3 , £ , 5 ) . ©

0-8412-0404-7/78/47-067-129$05.00/0


130


TABLE I. Number of animals and production Developed countries Cattle Buffalo Sheep Goats Pigs Chickens Ducks Other

Meat Milk Eggs Wool

3

1

Developing countries

Centrally planned countries

285,189 1 351,329 15,863 174,305 1,618,783 11,122 15,240

660,658 507 432,348 307,181 110,137 1,690,655 78,931 74,447

202,588 551 258,396 70,814 366,104 2,335,629 49,197 35,070

53.33 207.9 11.2 1.41

20.05

36.73

0.57

0.61

1 El-Shazly and Naga (1) 2 Animal numbers i n thousands 3 Production i n million tons

TABLE I I . Meat and milk supplying minimum animal protein ^ requirements for the world population i n 1970, 1985 and 1990

Minimum animal protein requirements (thousand tons/day) Projected world meat consumption (million tons) Protein consumption from meat/day (thousand tons/day) Projected world milk consumption (million tons) Protein consumption from milk/day (thousand tons/day) Protein consumption from meat and milk/day (thousand tons/day)

1970

1985

1990

105

142

154

107

168

197

58

92

108

389

532

597

37

51

57

95

143

165

1 El-Shazly and Naga (1)


8.

CHALUPA

A N D ΒA I L E

Diets for Food-Producing Animals

131

Diets for food-producing animals also routinely contain addi tives which do not supply nutrients. Agents which control disease, increase growth rate and/or improve feed efficiency include anti biotics, antibacterials, anabolics and chemicals which control specific pathways of rumen fermentation. Compounds which improve handling, processing properties and acceptability are also u t i l i z e d (6). Diet Formulation The objective i n formulation i s to translate knowledge about nutrients, feed ingredients and animals into diets that w i l l be consumed i n sufficient quantities to provide nutrients for main tenance plus production at least cost. Mathematical principles are relatively simple but because of the multitude of manipula tions required to provide exact amounts, minimums, maximums, ranges or ratios of nutrient linear programming method models being developed to describe entire production systems for mulate diets based upon sex, age, breed, frame size and i n i t i a l flesh condition of animals, feed additives and growth stimulants used, and environment; they simulate use, grade and price d i f f e r entials and make economic projections. Information obtained can be used to select the most profitable type and weight of animal and evaluate the economics of alternative feeding systems (7). Feed Ingredients Animals are often c r i t i c i z e d as consumers of food grains which could be consumed directly by humans. However, grains are fed when they are economical sources of nutrients and when prices increase, less grain i s fed to livestock and more i s sold directly (8) . In 1974, 50% of the world's grain production was consumed directly by humans, 40% went into animal feeds and the remaining 10% was used for industrial processes, seed and other purposes (9) . Seventy-five percent of the feed grains produced i n the united States was fed to livestock but this accounted for only 27% of feed consumed (9) because nutrients were also provided by forages, residual materials from the manufacture of human foods, crop residues, animal wastes and pure chemical forms of nutrients. Ruminants i n particular provide a mechanism to u t i l i z e nutrients from the 64% of the world's agricultural land suitable only for forage production (10) and the large reservoir of carbon i n crop and industrial by-products (11). The Ralston Purina Company Arkavalley Farm at Conway AR i s an excellent example of food production u t i l i z i n g limited quantities of conventional food grains and protein supplements. During 1974, 1414 milking cows consumed daily 30 kg sorghum silage and 14 kg of the grain mixture i n Table III and produced an average of 21 kg milk daily (12). Sixty-nine percent of the ingredients i n the


132


TABLE III. Composition of grain rations fed to milking cows at Arkavalley Farm during 1974Λ Ingredient

%

Corn Milo Rice bran Soybean hulls Wheat middlings Cottonseed meal Soybean meal Brewers grains Urea Fat Molasses Vitamins and minerals

1.04 18.78 20.75 24.14 5.47 12.46 6.02 3.48 ·$ -47 1.92 4.8 5

Kertz and Everett (12) concentrate mixture (i.e. rice bran, soybean hulls, wheat midlings, cottonseed meal, brewers grains, urea, fat and molasses) are not suitable for human consumption or processing costs to make them suitable are prohibitive. Of the remaining 31%, com, soy bean meal, and milo could be converted into food for humans but milo i s not used for human food i n the united States. With beef cattle, 1 kg of beef protein can be produced with 3 kg or less of feed grain plus o i l seed meal protein (8). This i s a birth.to* market calculation which takes into account that perhaps 80% of the beef animal's lifetime diet i s forage and that about 75% of the beef i s produced before the animal goes on a grain diet. This ratio can be narrowed further i f some nonprotein nitrogen i s used as a protein concentrate extender (10). Voluntary Feed Intake and

Production

The maintenance of an energy balance i n animals requires that the metabolizable energy available to an animal be similar to the energy demands (or energy loss plus deposition). Feed i s the source of metabolizable energy and, therefore, a high correlation usually exists between feed intake and the rate and efficiency of production. What i n fact i s being regulated or which are the de pendent and which are the independent variables of the energy me tabolism systems of an animal are not always apparent. Clearly, production of milk or growth rate can be reduced by limiting feed intake, but either can increase feed intake. For any one diet there probably i s a maximum feed intake for an animal, under a single set of conditions, due to both physical and metabolic


8.

CHALUPA AND ΒAILE

Diets for

Food-Producing Animds

133

factors. Various interrelated factors determine how near this maximum intake an animal w i l l voluntarily consume. There i s a commonly held belief that animals fed ad libitum eat a l l they "can". But i s i t implied that there are some physi cal limitations to eating more, or rather physiological and/or psychological limitations? Several species have been shown to be capable of digesting and metabolizing substantially more feed than that voluntarily eaten. For example, chicks force-fed for 15 days so that their intake was 170% of the control group grew 50% faster and retained nearly 40% more of the feed-derived energy i n their carcass than did the controls (13). Therefore, the control chicks were eating at a level that did not approach their physiological capabilities. It i s a common observation that those groups of animals which eat more of a specific ration gain more rapidly and e f f i c i e n t l y . As i l l u s t r a t e d i n Figure 1, growth rate i s proportional to the net energy available for growth remains constant. Sinc established by animals reaching a certain weight range or f i n i s h and not a set number of days, the cost of production i s reduced for animals with a greater proportion of net energy available for growth as a result of their greater feed intakes. As shown i n Figure 2, the cost of net energy for maintenance decreases (for the period required for a 200 kg gain i n a steer) as the propor tion of net energy for growth i s increased to that for maintenance, which could result from increased feed intake. Therefore, less feed i s required for the production of the same quantity of meat from an animal which i s eating more and making a greater propor tion of the total net energy available for growth on any one day, because the steer needs to be maintained fewer days. The controller for feeding i s subject to modulation by several relatively independent physiological systems as i l l u s t r a t e d i n Figure 3. Feeding behavior i n a wide range of environmental con ditions appears to operate quite independently of the body temper ature regulatory system. But under heat stress this system can apply considerable pressure to the controller of feeding behavior resulting i n reduced intake. Similarly, the regulator for body water balance plays a very passive role except under conditions of dehydration which places restrictions on feed intake. A third modulator of feeding behavior encompasses a wide variety of sen sory inputs with many roles. Briefly, external sensory cues are important i n the selection or aversion of possible foods. Also, they very often potentiate hunger drives or the inhibition of sa tiety and thus participate i n precipitating slight shifts i n moti vation to produce meal patterns — feeding-no feeding conditions. The internal condition modulation i s frequently the only apparent cause of the hypophagia associated with most pathological states. The remaining system i l l u s t r a t e d i n Figure 3, the energy bal ance regulator, i s theoretical, but there i s substantial circum stantial evidence that i t may exist. This regulatory system i s


FLAVOR CHEMISTRY OF ANIMAL FOODS 400 KG STEER

*

1 8

m

ο

I

i «

m 125

I

1.0 g

ζ

I

m

15

Figure 1. The dependence of rate of gain on the proportion of the net energy available for growth (G). These refotionships assume a 400-kg steer is receiving the same diet but in increasing amounts relative to that required for maintenance (M).

125

150 175 200 FEED INTAKE AS % OF MAINTENANCE

Figure 2. The cost of growing a steer from 300 to 500 kg when in creasing feed intakes refotive to maintenance are maintained. The requirement for net energy for the gain (G) of the 200 kg remains constant, but the net energy required for maintenance is reduced as the number of days required for the gain is reduced.


8.

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135

INTERACTION OF BODY REGULATORY SYSTEMS AND THE CONTROLLER OF FEEDING

KINETIC ENERGY REGULATORY SYSTEMS (BODY TEMPERATURE REGULATOR)

WATER BALANCE REGULATORY SYSTEMS

POTENTIAL ENERGY REGULATORY SYSTEM (BODY ENERGY DEPOT REGULATOR)

CONTROLLER OF FEEDING BEHAVIOR

INTERNAL AND EXTERNAL ENVIRONMENTAL QUALITY SENSORY SYSTEMS

Oklahoma Veterinarian

Figure 3. The dependence of the controller of feeding behavior on several physio logical systems. Body temperature and water balance regulatory systems apply Little pressure on the controller of feeding behavior except when either reach much imbalanced conditions, e.g., heat stress, water dehydration. The sensory systems are espe cially important in initiating a meal and in selecting and avoiding food stuffs. There is strong circumstantial evidence for an energy balance regulatory system which modu lates feeding behavior (14).



136

thought to be the reason body weight (or body energy content) remains rather constant i n a variety of environmental and nutritiona l conditions. I t applies pressure on the controller of feeding behavior so that the summation of the energy derived from the meals per day or per week closely matches the energy demands (15). Therefore, the concept of a relatively independent energy balance regulator requires that there be an interface with the relatively dependent controller for feeding. Chemical Senses and Feeding The chemical senses are composed of stimuli from the external environment which act on specialized c e l l s . They are commonly divided into three classes: 1) olfaction, 2) gustation, and 3) the common chemical sense. Olfaction permits reception of distant airborne v o l a t i l e substances often at very diluted concentrations while gustation generall ulant source. Irritant chemical sense and involve l i t t l e s p e c i f i c i t y . Food-producing animals u t i l i z e a sniffing behavior often associated with the apprehension, selection and prehension of feeds. The importance of odor i n food selection by sheep was investigated by offering control and bulbectomized sheep food from control containers versus containers that had been adulterated with acetic acid, camphor or iodobenzene (Table IV). Bulbectomized sheep selected approximately equal quantities of food from each container whereas control sheep only consumed about 25% of their 60-minute intake from adulterated containers (31). Taste i s very species dependent and seldom i s predictable based on the experiences and preferences of man. While we c l a s s i fy our tastes as sweet, sour, b i t t e r and salty, animal behaviors in relation to taste are better described as evidence of preferences, aversions, or indifference (19). In studies with cattle, sheep and normal and pygmy goats with solutions representative of the four c l a s s i c a l tastes, cattle discriminated sweet, salty and sour tastes at lower concentrations than the other species. Catt l e , however, showed the poorest discriminating behavior with b i t ter tastes and also tolerated the b i t t e r taste at the greatest i n tensity (20). As suggested above, the chemical senses play several roles i n the selection of feed and the control of feed intake. The animal can sometimes be induced to adjust i t s feeding behavior by changing sensory qualities of i t s feed. The aversive sensory qualities of certain feeds can be masked to allow economical u t i l i z a t i o n of the feeds. For example, i t has been shown that sheep w i l l eat aversive feeds i f a local anesthetic, e.g. carbocaine, has been added to the feed (16). In studies with taste modifiers on responses of pygmy goats to sucrose and quinine hydrochloride (17), gymnemic acid and monosodium glutamate reduced sensitivity to sweet and b i t t e r substances whereas inosinic acid enhanced


8.

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137


TABLE IV. Percent feed eaten after sixty minutes from treated containers by control and bulbectomized sheep given a choice between treated and control containers.

Treatment

Control No. Sheep

Acetic acid (Moles/kg feed) .21 .05

%

Bulbectomized No. Sheep

5 1

3

2

12 8

30+3.8 41+6.2

6 5

± · * 51+5.7

Camphor

12

22+6.1

6

58+5.6*

Iodobenzene

12

25+8.9

6

63+7.5

2

1 Baldwin, McLaughlin an 2 A gauze saturated with either a 33% camphor solution or iodoben zene was wiped around the inside top of the container to form a 2 to 5 cm ring. Significantly different from control sheep at P_ 4 g/Kg, 20) has also been found to inhibit feeding i n laboratory rats (21). The results of this study indicated that P. b a i l e y i possesses a mechanism for detoxifying simmondsin that the others lacked. It i s also interesting to note that earlier work by Rosenzweig and Winakur (22) suggested a direct connection between the distribution of jojoba and that of P. b a i l e y i . Thus the jojoba plant completely protects i t s seeds from three potential predators by employing a toxic principle (simmondsin). This i s not a static system however, for one mammal, at least, i s equipped with a mechanism to detoxify simmondsin and can u t i l i z e jojoba seeds. Cook et a l . (23) examined the relationship between the mourning dove (Zenaida macruora) i n California and one of i t s favorite foods, the seeds of the dove weed (Eremocarpus setigera). They reported that two types of seeds are produced by this plant:


10. ROGERS

Repellents to Protect Crops

153

mottled seeds that are highly palatable; and grey seeds that are not palatable. They further report that water extracts of grey seeds contain a chemical that i s toxic to goldfish. Water extracts of mottled seeds do not contain the toxic principle. This material they assumed, i s what affects the palatability of the grey seeds to doves. Figure 1 i l l u s t r a t e s the extreme selectivity of mourning doves given a mixture of mottled and grey seeds for a 30 minute feeding t r i a l on each of 5 consecutive days. Field data (23) also demonstrate an increase i n proportion of grey seeds (3.8% vs. 30.2%) i n two samples collected about 6 weeks apart from a f i e l d population of Eremocarpus. The increase was attributed to doves selecting the more palatable mottled seeds. This unpalatability of grey seeds i s accompanied by a reduced v i a b i l i t y of the seeds. The polymorphism has a genetic base. Its phenotypic expression i s apparently induced by adverse environmental conditions and senescence that cause a shift to the "grey syndrome". Along with reduce produces almost complet the metabolic cost of producing the unpalatability i n grey seeds causes a decrease in v i a b i l i t y that i s outweighed by the advantage of protection from doves i n areas where seed prédation i s important. Sherburne (24) studied the feeding patterns of a number of frugivorous birds on several species of fleshy-fruiting plants in New York. He concluded that unripe f r u i t s were actively avoided by birds. The avoidance was hypothesized to result at least i n part from the presence of toxic secondary compounds i n the unripe f r u i t and the disappearance or diminished concentration of these same compounds from the ripe f r u i t s . This was directly demonstrated for the f r u i t of one of the plants he studied (Rhamnus cathartica). Another polymorphic population, that of the wild ginger (Asarum caudaturn)was studied by Cates (25) i n western Washington. He noted that populations of this plant are polymorphic for growth rate, seed production, and palatability to a native slug (Ariolimax columbianus). In sites where slugs were abundant the unpalatable form (characterized additionally by fewer seeds and later time of flowering) predominated, whereas the converse was true i n those sites where slugs were less common (Figure 2). These data led Cates (25) to hypothesize that i n areas where slugs were not abundant those plants that allocated more of their energy into growth and reproduction would be at a competitive advantage. However where slug prédation was greater, those plants emphasizing the antiherbivore characteristic (unpalatability) would be selected for. The foregoing examples lead to several conclusions about the antiherbivore characteristics of plants that hold important implications for the development and use of repellents. They are: (1) Part of the protected plant must be eaten; (2) not a l l plants have u t i l i z e d the same secondary substances; (3) not a l l secondary



Figure 1. The responses of mourning doves (6) to mixtures of 30 mottled (M) seeds and 15 grey (G) seeds. Drawn from data presented by Cook et al. (23).

HABITAT OF:

Figure 2. Relationship between the number of slugs and flowering time of wild ginger (Asarum caudatum) in the habitat of palatable and unpalatable morphs. Redrawn from Cates (25).


10.

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155

substances are effective against a l l herbivores; (4) toxicity or some other adverse physiological effect i s an important aspect of each plant-developed repellent; (5) the substances are not necessarily bad tasting upon f i r s t contact; and (6) a l l require a behavioral adjustment on the part of the herbivore. Poison Avoidance by Animals The necessary behavioral response to a repellent has received l i t t l e attention i n the literature of repellency. On the surface i t seems obvious that the required response i s a cessation of feeding on the protected source. The physiological-psychological mechanism to achieve this result i s less obvious. Most species have developed a behavioral mechanism with which to cope with toxic substances i n their food. A brief reading of the vast literature concerning the use of toxicants against rats demonstrates this phenomenon rat poisons i s that thos toxicant refuse to consume any more of the poisoned food (26). This behavioral response, a problem i n lethal control, i s exactly the behavior we are trying to induce with the use of repellents. This adaptive behavior (bait shyness) has been exhaustively studied as conditioned taste aversion by psychologists. Rozin and Kalat (27) have summarized much of the exhaustive literature, principally for the rat, and have described how the rat handles the problem of food selection: "...A rat becoming sick at a garbage dump [where he was poisoned]....may have eaten a few different foods. He knows i t was a food that made him sick and can discount any familiar safe foods. With the capability of forming associations over long delays, he i s now l i k e l y to associate his illness with the last relevant thing or few things he ate over the last few hours....(p. 478)". Thus, the taste of a food that made an animal sick i s subsequently avoided-a conditioned aversion i s formed. Though i t seems probable that most i f not a l l pest species are capable of learning conditioned aversions i n the laboratory, the conditions under which this most readily occurs would not necessarily be present i n crop depredation situations. The depredating species i s required to form an aversion to a familiar food-aversive agent combination. One might expect the learning of the aversion to be more d i f f i c u l t given the importance of novelty i n taste aversion learning (27). It has, however been shown (28)that at least one crop depredating species, the redwinged blackbird (Agelaius phoeniceus), i s able to accomplish this with relative ease. The possible decrease i n response because the treated food has previously been considered as "safe" might be somewhat ameliorated by the fact that the aversive agent (repellent) would not usually be present i n one of a vast array of foods. Thus the repellent would be expected to add a novel or unfamiliar taste to the familiar food. A great proportion of crop


156


depredations occur i n nearly monoculture situations (e.g., corn, r i c e , large f r u i t orchards) where, before the onset of the adverse post-ingestional effect, the target species would be expected to have consumed only a very limited number of foods, and possibly only the protected crop with the repellent material added, thereby simplifying the problem of associating the illness with a particular food. Mammalian species seem to be able to form conditioned aversion^ most readily to gustatory cues and rely upon other foodrelated cues (visual, olfactory) to a lesser extent (27). Evidence indicates that at least one bird, the bobwhite quail (Colinus virginianus), may form aversions more readily to visual than to gustatory cues (29). Nevertheless bobwhites, and more than l i k e l y other avian species, were able to u t i l i z e gustatory stimuli as cues to adverse post-ingestional effects. The question might be asked whether the reaction to a toxic material in the food i effectiv t alterin food habit than the other possibl bad-taste effect. Two line sugges this question. F i r s t , and most powerful, i s the evidence, d i s cussed above from plant evolution. The plants have emphasized defenses that adversely effect the physiology of their vertebrate predators, they have not frequently used taste stimuli except as cues to toxic events. Second, i s an experiment (30) that directly compared the effectiveness of toxic and non-toxic materials at altering the feeding behavior of red-winged blackbirds. In this experiment several materials were compared i n three, two-choice tests where the palatability of the alternative to the treated food was varied from highly palatable through mildly avoided to highly offensive. By measuring the time the birds took to transfer feeding to the alternative i t was possible to examine the motivating strength of the various repellent stimuli (Table I). In these tests i t was determined that the only materials consistently effective i n altering the feeding behavior were those that confronted the bird with a choice between illness for continuing to eat the treated food, and the alternative bad-tasting but toxicologically harmless diet. The data from that experiment supported the very important contention of Alcock (31) who suggested that except as signals to toxic or emetic effects, negative gustatory cues from prey are of l i t t l e significance i n determining food preferences of wild animals. Applications of Conditioned

Repellency

The idea of using the conditioned aversion approach to the development of chemical repellents i s not new. It was l i s t e d by Neff and Meanley (1) as one of thé possible types of repellency, but was considered in their review as distinct from "true repellency". Tevis (32) after a study using 1080 (sodium fluoroacetate) as a poison on Douglas-fir (Pseudotsuga menziesii) seeds to


10.

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157

protect them from rodents suggested that one of the advantages of bait shyness as a repellent response was that i t created a population of non-seed-eating rodents that continued to be r e s i dent i n the area. Their continued presence did not allow the great influx of migrants that commonly occurs after lethal control campaigns. The p o s s i b i l i t y of conditioned aversion as a mode of repellent action has also been raised i n passing by others (e.g., 32, 34, 35) but generally was not pursued u n t i l recently. Lately, the concept has received increased attention, possibly because of the failure of other approaches, or because of the increase i n our knowledge of the principles of taste aversion learning. Currently conditioned aversion as a repellent response i s being used to manipulate the food habits of three diverse depredating groups (wild canines, seed-eating rodents, and f r u i t and seed-eating birds). I t i s interesting that i n these three cases the conditioned response i s the mechanism being exploited, but that in each situatio different route. The f i r s t of these cases, while not involving a botanical "crop" i n the traditional sense illustrates one route of approach. Coyote (Canis latrans) prédation upon sheep and lambs in the Western U.S. i s locally severe. With the controversy over destructive measures of coyote control and the general removal of the poisons 1080 and strychnine for use against them, new approaches are being sought to control coyote prédation where i t occurs. John Garcia (a pioneer i n the development of knowledge of conditioned aversion i n the f i e l d of psychology) and several of his associates, recognizing the ubiquity of the conditioned response, tested this mechanism directly under f i e l d and laboratory conditions as a means of controlling coyote prédation. In the restricted laboratory environment they (36) discovered that coyotes could learn after one t r i a l with (toxic) lithium chloride - treated meat to avoid the flesh of that prey. They reported that a few t r i a l s with treated lamb or rabbit flesh specifically suppressed the attack upon the averted prey. A limited f i e l d test of this phenomenon using lithium chloride-treated packets of sheep meat and treated carcasses indicated to them that their treatment had caused a 30-60 percent reduction in sheep k i l l e d by coyotes on the 3000 acre sheep ranch where the test was carried out (37). The approach taken by this group involved the recognition of the strength of the conditioned response and employing i t to help solve a specific problem. They have verified that feeding can be manipulated by this means i n the laboratory and are attempting to establish conditions that w i l l control prédation in wild populations. Endrinl was used for a number of years i n combination with 1.

Use of chemical, trade, or company names does not imply endorsement by the Federal Government.


158


tetramethylthiuramdisulfide (TMTD) as a seed treatment to deter rodent feeding on conifer seeds. This formulation (which may act by the conditioned route) i s no longer acceptable because of i t s nonselectivity, high toxicity, and persistence i n the environment (38). The search for a replacement for endrin focused upon mestranol {3-methoxy-19-nor-17a-pregna-l,3,5 (10)-trien-20-yn-17o l , Syntex Corp.}, a synthetic estrogen used as a reproductive inhibitor (39). Mestranol was selected as a candidate repellent because when used as a reproductive inhibitor i t produces a conditioned aversion i n the target rodents (40, 41). Registration of this material (2% w/w) as a repellent on conifer seeds i s pending. We have here a case of a material that was a failure for i t s intended use being recognized (because of "bait shyness") for another. Finally, the long screening search for a bird repellent has l e f t us for a moment with a single material, methiocarb. Methiocarb {3,5-dimethyl-4(methylthio)pheno Chemagro Division, Bayche repellent for use on corn seed and ripening cherries. Its success was shown to be due i n part to the fact that i t acts as a conditioning repellent (30). Because of the potential of methiocarb as a broad-spectrum bird repellent i t has received much attention aimed both at improving i t s usefulness and as a model conditioning repellent for studying conditioned aversion i n depredating birds (30, 28). Work has indicated that the conditioned response acquired to i t by red-winged blackbirds persists i n the laboratory for periods up to 16 weeks (Fig. 3). Additionally, this species forms an aversion directly to the methiocarb and can recognize the material on other foods as well as i t s absence from the original food (Table II). Behavioral observations of common grackles (Quiscalus quiscula) feeding i n newly planted corn plots indicate that these birds quickly learn to discriminate between treated and untreated seed (42). Under these f i e l d conditions the observed birds consumed a significantly higher proportion of untreated seeds (Table III) while not being repelled from entering the treated area thus allowing them to remain and perform any beneficial a c t i v i t i e s . Thus research to date with methiocarb indicates that i t i s effective because i t possesses a taste that can be used as a cue to i t s toxic post-ingestional effects. In the three examples presented, the conditioned aversion response played the leading role. In the f i r s t , i t was recognized that the response i s probably the most effective at manipulating the food habits of a wild carnivore. In the second, the problem associated with this response ("bait shyness") i n getting adequate acceptance of a treated bait when using a reproductive inhibitor led to i t s use as a repellent. In the third, extensive screening efforts for a bird repellent identified one material, methiocarb,


10.

ROGERS


159

Table I. Average number of hours required by male red-winged blackbirds to learn to avoid several repellants. Numbers i n parentheses refer to numbers of birds of 18 tested that responded (sucrose octaacetate) or survived (methiocarb, L i C l ) . NR, no response. From Rogers (30). a

Compounds^ LiCl

Choices

SOA

Treated vs. untreated mash

2.8 (18)

2.4 (18)

2.7 (18)

Preferred vs. nonpreferred (corn or rice)

3.2 (18)

6.6 (13)

10.0 (12)

Mash vs. DMA checkerettes

NR

9.1 (12)

11.4 (11)

(0)

Methiocarb

êans not underlined by the same line are significantly different from each other by Duncan's New Multiple Range Test (P < 0 . 0 1 ) . PRETEST^ 60 5550 Ο

45·

Ô 40ui

iV\

ui

35-1

u

S0«

ui

2

GBFC AFTER AFTER REST 7

y S

V

20 METHIOCARB AFTER REST ιο

I I 2 0ΑΥ Κ

4

8 WEEKS —

16

-J

Figure 3. Feeding responses of six groups of nine male red-winged blackbirds at various intervals after formation of a conditioned aver sion to 0.07% methiocarb. Top curve; feeding on untreated food before training. Middle curve; feeding on untreated food after the rest interval (1 day, 1, 2, 4, 8, 16 weeks) before retesting with methiocarb. Bottom curve; feeding on treated food after the rest interval. GBFC refers to the normal diet. From Rogers (28).


160


Table II. Feeding response i n a 1-min exposure of eight male redwinged blackbirds to untreated foods and foods treated with 0.07 percent methiocarb. The treatments are arranged i n order of presentation from top to bottom (28).

Treatment

Time spent feeding

Pretest untreated GBFC

57.9 + 2.1* +

First exposure methiocarb i n GBFC

59.5 + 0.5*

Methiocarb i n GBFC after training

5.1 + 3.4

Untreated GBFC

45.5 + 7.0 +

Untreated rice

48.5 + 7.0* +

F i r s t exposure methiocarb i n rice 17.4 + 3.9 A l l means not marked with the same symbol are significantly d i f ferent from each other (Duncan's New Multiple Range test (P = 0.05). GBFC = normal diet.


10.

ROGERS

161


Table III. Feeding a c t i v i t i e s of grackles that entered both the treated and untreated plots i n the experimental corn seed planting. Number of birds i n parentheses (42).

b

Seconds/row

Total No. seeds eaten Seconds/seed* Total No. seeds dropped

a

5.6±0.9 15 29.2±2.8 3

7.1±1.0

6.3±0.8 (47)

59

74 (29)

24.0±7.7 28.1±2.7 7

10

1975 - 24 untreated and 12 rows of seed treated with methiocarb. 1976 - 18 untreated rows and 18 treated rows.

b

Means + SEM.


162


that i s effective because of i t s mode of action - the conditioned aversion. It may be inferred from the preceding discussion that I am advocating the use of naturally occurring plant chemicals as bird repellents. This i s an attractive p o s s i b i l i t y that has been suggested before (cf. 24). However, because of the high, broad spectrum toxicity and chemical l a b i l i t y of many of the plant secondary substances i t might not prove a practical approach. Plants, along with developing secondary substances have often developed an e f f i c i e n t delivery system (e.g., vacuoles and vesicles) that isolates the chemical from the environmental conditions that might promote rapid degredation. For this reason the practical aspects of treating a crop might make this approach unfeasible. In the e a r l i e r discussions of the development of chemical defenses against prédation by wild ginger (25) and dove weed (23) i t w i l l be recalled that whereas these chemicals conferred protection from prédation to the plants. In both were at a competitive disadvantage when prédation was absent. Though data are extremely limited, three unpublished tests of methiocarb as a bird repellent (43, 44^ 45) indicated that under conditions pertaining i n New York, Delaware, and North Carolina in 1975 methiocarb, though reducing the number of corn sprouts damaged by birds, did not increase the sprout population that emerged and survived the period of vulnerability to bird damage. In other words methiocarb reduced bird damage, as measured by observations of missing and damaged sprouts, but also may have carried with i t some phytotoxic effects. Thus, i t may be that, as with some plants that have developed their own chemical defenses, the addition of a chemical repellent to a crop may only be advantageous under conditions of high levels of herbivore prédation. Conclusion Whatever mode(s) of action the ultimate repellents for protection of agricultural crops from vertebrate pests emphasize i t would be important to reiterate the lessons learned from the chemical defense of plants against herbivores and to restate them in terms of man-developed repellents. F i r s t , i t i s reasonable to expect at least a low level of damage during the conditioning period of the pest population - 100 percent protection i s not a reasonable expectation. Second, because of differences i n the crop, a repellent that i s effective at protecting one crop w i l l not necessarily protect others - physical characteristics of the crop may dictate differing rates of consumption, or techniques of consumption (e.g., hulling of topically treated seeds by the depredating species). Third, i t i s very unlikely that any one repellent w i l l be effective against a l l depredating s p e c i e s — i t i s unreasonable to expect a panacea because of differences i n


10.

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163

behavior and physiology of the species involved. Fourth, a l l the presently viable vertebrate repellents (as defined earlier) involve as an important part of their mode of action some adverse effects upon the physiology of the target species - i t i s l i k e l y that future repellents w i l l possess the same components. F i f t h , the effective repellents are not necessarily bad-tasting—the pest learns to associate the taste with an adverse physiological effect. Sixth, the use of a repellent demands a behavioral response from the target animal - this response i s an alteration i n feeding behavior. Seventh, any repellent i s l i k e l y to be most effective when adequate alternative foods are available. Conditioned aversion i s the mode of action that most l i k e l y w i l l produce the required response. The response, of course i s an alteration of food habits. As a whole these points should make i t obvious that there w i l l be no panacea repellent and only through a coordinated series of studies on a l l aspects o w i l l appropriate repellent account the behavior, physiology, and ecology of the specific problems to understand the nature of the problem. We must understand the problem before effective repellents can be developed. Acknowledgement I would l i k e to thank G. Beauchamp, R. Dolbeer, W. Jacobs, J. Linehan, and D. Senseman for helpful suggestions during the preparation of this manuscript. Literature Cited 1.

Neff, J. A. and B. Meanley. Progress Report No. 1. Denver Wildlife Research Center, Denver, Colorado (1956). 2. Welch, J. F. J. Agr. and Fd. Chem. (1954) 2:142-149. 3. Besser, J. F. and J. F. Welch. Trans. 24th North American Wildl. Conf. (1959) 24: 166-173. 4. Armour, C.J. Forestry Abstracts (1963) 24: xxvii-xxxviii 5. Dambach, C.A. and D.J. Leedy. Trans. 14th N. American Wildl. Conf. (1949) 14: 592-603. 6. Ehrlich, P.R. and P.H. Raven. Evolution (1964) 18: 586-608. 7. Brower, L.P., J.V. Brower, and J.M. Corvino. Proc. Nat. Acad. Sci. (1967) 57: 893-898. 8. Fraenkel, G.S. Science (1959) 129: 1466-1470. 9. Fraenkel, G.S. Entomologica experimentalis et applicata (1969) 12: 473-486. 10. Hsiao, T.H. Entomologica experimentalis et applicata (1969) 12: 777-788. 11. Whittaker, R.H. "The Biochemical Ecology of Higher Plants". In: E. Sondheimer and J.B. Simeone (Eds.). Chemical Ecology. Academic Press, New York, 1970.


164 12. 13. 14.

15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34.

35. 36. 37. 38. 39. 40. 41. 42.


Whittaker, R.H. and P.P. Feeny. S c i . (1971) 171: 759-770. Freeland, W.J. and D.H. Janzen. Amer. Nat. (1974) 108: 269-289. Dethier, V.G. "Chemical Interactions between Plants and Insects". In: E. Sondheimer and J.B. Simeone (Eds.). Chemical Ecology. Academic Press, New York, 1970. Gilham, M.E. J. Ecol. (1955) 43: 172-206. Jones, D.A. Nature (1962) 193: 1109-1110. Jones, D.A. Genetica (1972) 43: 394-406. B e l l , E.A. & D.H. Janzen. Nature (1971) 229: 136-137. Sherbrooke, W.C. Ecol. (1976) 57: 596-602. E l l i g e r , C.A., A.C. Waiss, Jr., and R.E. Lundin. J . Chem. Soc., Perkin Trans. (1973) I: 2209-2212. Booth, A.N., C.A. E l l i g e r and A.C. Waiss, Jr. L i f e Sci. (1974) 15: 1115-1120. Rosenzweig, M.L. and J . Winakur. Ecol. (1969) 50: 558-572. Cook, A.D., P.R. Atsatt 21: 277-281. Sherburne, J.A. Unpub1. Ph.D. dissertation (1972), Cornell University, New York. Cates, R.G. Ecology (1975) 56: 391-400. Rzoska, J. Anim. Behav. (1953) 1: 128-135. Rozin, P. and J.W. Kalat. Psychol. Rev. (1971) 78: 459-486. Rogers, J.G., Jr. Auk (1977): i n press. Wilcoxon, H.C., W.B. Dragoin, and P.A. Kral. Sci. (1971) 171: 826-828. Rogers, J.G., Jr. J . Wildl. Manage. (1974) 38: 418-423. Alcock, J. Anim. Behav. (1970) 18: 592-599. Tevis, J . , Jr. J . Mammal. (1956) 37: 358-370. Rediske, J.H. and W.H. Lawrence. Forest Sci. (1962) 8: 142-148. Teichner, W.H., R. Warranch, M. Lopiccolo and C. Campbell. U.S. Army Natick Labs (Natick, Mass.) Tech. Report. 70-69PR (1969). Omura, K., S.F. Takagi and O. Harada. Gunma J. Med. S c i . (1961) 10: 217-227. Gustavson, C.R., J . Garcia, W.G. Hankins, and K. Rusiniak. Science (1974) 184: 580-583. Gustavson, C.R., D.J. Kelly, M. Sweeney and J. Garcia. Behav. Biol. (1976) 17: 61-72. Radwan, M.A. Proc. 4th Vert. Pest. Conf. (1970) 4: 77-82. Lindsey, G.D., R.M. Anthony, and J. Evans. Proc. 6th Vert. Pest. Conf. (1974) 6: 272-279. Howard, W.E. and R.E. Marsh. J. Wildl. Manage. (1969) 33: 403-408. Marsh, R.E. and W.E. Howard. J. Wildl. Manage. (1969) 33: 133-138. Rogers, J.G., J r . and J.T. Linehan. J . Wildl. Manage. (1977) 31: i n press.


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43.

44.

45.

ROGERS


165

Linehan, J.T., C.R. Ingram and P.W. Lefebvre. Unpub1. report U.S. Fish and Wildlife Service, Patuxent Wildlife Research Center, Laurel, Md. (1975) 10 pp. Mitchell, R.T., B. Meanley, R.E. Matteson and C.R. Ingram. Unpub1. report U.S. Fish and Wildlife Service, Patuxent Wildlife Research Center, Laurel, Md. (1975), 10 p. Stickley, A.R., J r . and C.R. Ingram. Unpub1. Report, U.S. Fish and Wildlife Service, Patuxent Wildlife Research Ctr., Laurel, Md. (1975) 11 pp.

RECEIVED October 25, 1977.


INDEX Ants Area attractants, food odors as Armadillo L-Aspartyl-L-phenylalanine Astringents Attractant(s) coyote 2,94 feline 70,75 food odors as area Attractanc t odorant

A Acceptability of food, factors influencing 45 Acceptability test, conditions of the .... 49 Acceptance 149 Acetic acid 3,30,136,137 Acid(s) (see also specific kinds)

fraction Adaptiveness of wild birds and mammals, physiological an behavioral 2 Additives used toflavorfeeds 138 Additive, rice volatile 36 Aerobic bacteria in red fox anal sac secretions 81 Agents, empirical screening of flavoring 31 L-Alanine 2,3 Alarm 93 substance 99 Alcohol 46 Alkaloids 3,31,94,152 Altered taste threshold as a function of body state 48 Amino acids 118,124 2-Amino acids 113 D-Amino acids 2 Aminoethanol 94 5-Aminovaleric acid 83 Ammonium chloride 3 N-Amylamine 34 Anal sac 79 micro-ecosystem 80 secretions, aerobic bacteria in red fox 81 Angiosperms 151 Animal(s) behavior biochemical and physio logical aspects of 92 diets for food-producing 129 flavor research 1 foods, methology of behavioral testing associated with development in 43 poison avoidance by 155 protein requirements for the world population, meat and milk supplying 130 psychophysics 44 Aniseed 138 Antelope 21,93 ;

99 31 8 3 31 93 84,86 99 31 88

Β Bacteria in red fox anal sac secretions, aerobic 81 Bacterial action and chemical signalling in mammals 78 Bait additives 31,32 Bait shyness 158 Bases fraction 70 Bears 100,102 Beetles, carrion 100 Behavior, biochemical and physio logical aspects of animal 92 Behavioral adaptiveness of wild birds and mammals 21 events exhibited 72,73 influences 27 studies 86 testing associated with development in animal foods, methology of .. 43 variables 43 p-Benzoquinone 30 Berylium chloride 3 Bioassay of urine fractions 67 Biochemical aspects of animal behavior 92 Bird(s) 14,22,152 food preference behavior in 21 pest 30 physiological and behavioral adaptiveness of wild 21 repellent 158 (methiocarb) 30 wild 29 Bitter stimuli, responses of mammals to natural 6

167


168


Bitter (continued) substances 8 (sucrose octaacetate) 9 threshold in cancer patients 48 Black-tailed deer 36,78 Blood glucose-homeostasis mechanism 24 Blood glucose, level of circulating 25 Blowflies 12 Bodily constitution 44 Body state, altered taste thresholds as a function of 48 surfaces of living animals 78 wisdom 47 Boll weevil 95 Br0nsted acid 121 Brucine 3 Buckwheat 62 Buffalo 13 N-Butyldiethanolamine 3 Butyric acid 3 Butyryl choline chloride 108, 111, 112 Buzzards 100

Caecotrophe 28 Caffeine 3 Caloric regulation 26 Calorically balanced diets 25 Calves, dairy 139 Camphor 96,99,136,137 Canadian peas 46 Cancer patients, bitter threshold in .... 48 Canned fabrication 146 Canned food flavor 32 Capsicum 138 Carbocaine 136 Carbohydrates 29 Carbonyl compounds 119 Carnassials (shearing teeth) 103 Carnivore 4 to amino acids, taste responses of 124 fungiform papillae taste system 121 heterocyclic stimuli in the 125 in nutritional ecosystem 103 taste systems,flavorchemistry of ... 102 Carrion beetles 100 Cat(s) 8,14,36,46,79,93,102 chemosensory neural groups in the geniculate ganglion of 108 civet 100 domestic 6,9 fungiform papillae taste systems .106,110 geniculate ganglion chemoresponsive units of 107

Cat (continued)

group, molecular circuitry associated with receptor activation by a 123 skulls 103 taste neuron responses to compounds infleshfoods 118 taste preferences 4,10 wild 5 Catnip 99 Cattle 8,93,130 Cavy 5,8,10,14 Cereal flavor 32 Change-over-delay (C.O.D.) 61 Chemical fractionation 67 fractions from estrus urine attractive to the coyote 66 input signals 120 signalling action and 78 stimulus factors in carnivore fungiform papillae taste systems 121 signals, microbially generated 78 Chemoreception 23,105 Chemoresponsive neural groups 106 Chemoresponsive units, cat geniculate ganglion 107 Chemosensory neural groups, the geniculate ganglion 108 Chenopodium vulvaria

Chickens

84

2,12,24,26,29,30,130,133

Chloride salts ( see also specific kinds )

Chloroethane 99 Chromatogram of acids fraction 75 Chromatography, gas-liquid 70 Circadium rhythms 25 Circuitry associated with receptor activation, molecular 123 Citric acid 3,6,36 Civet cats 100 Clostridium perfrigens

84

Cod liver oil 34 Column description 97 Component identification 67 Compounds and oil yield with selected grasses 97 Concurrent reinforcement schedules .58,60 Conditions of the acceptability test ... 49 Consummatory effort, principle of least 50 Control, food odors as area attractants to rodents for 31 Controller of feeding behavior on physiological systems, dependence of 135


29

169

INDEX

Corn oil 34 Corn seed planting 161 Cost of growing a steer 134 Coumarins 3 Cows, grain rations fed to milking 132 Coyote(s) 22,82,93,102,157 attractant(s) 84, 86 chemical fractions from estrus urine as a 66 fermented egg product as a 86 estrus urine acids, methyl esters in .. 76 test area 68 Creatine 118 Creatinine 118 Criteria for making food available 59 Crops from vertebrate pests, repellents to protect 150 Cyanide 22 2-Cyanomethylenecyclohexyl glucoside (simmondsin) 15 Cyclamate(NA) 3 L-Cysteine 10 D Death 93 Deer 93 black-tailed 36,78 musk 99 Defense 93 of plants, chemical 151 Demethyl disulfide 79 Denatorium benzoate (bitrex) 3 Diazomethane 67 Diet(s) calorically balanced 25 for food-producing animals 129 formulation 131 spiced 31 Dihydroethylaniline 34 Dipeptides 117 Diphosphate nucleotides 119 Discharge patterns of cat geniculate ganglion chemoresponsive units .. 107 Dog 79,83,102 chemosensory neural groups in the geniculate ganglion of the 108 foods, semi-moist 142 fungiform papillae taste systems .... 110 skull of 103 Domestication, effects of 26 Dove, mourning 152,154 Dove weed, seeds of the 152 Drugs 48 Ducks 130

£ Ecological influences, behavioral 27 Ecosystem, carnivores in nutritional .. 103

Endrin 158 Energy available for growth, dependence of grain rate on net.. 134 Enhancers, sweetness 31 Egg product as a coyote attractant, fermented 86 Elk 21 Environmental fermentative scent sources 84 origin, microbially generated chemical signals of 78 scent sources 78 Escherichia coli

Essential oils of plants Estrus cycle Estrus urine acids, methyl esters of coyote attractive to the coyote, chemical fractions from Ethy Ethyldiethanolamine Ethyl-terf-butyl ether Experience in food selection modification offlavorpreferences by individual previous Extracted compounds, intensifying flavor with

80

99 25

76 66

34 99 15 11 48 33

F Fabrication canned 146 dry 146 product 145 semi-moist 146 Factors influencing the acceptability of food 45 Fat 137 Fatty acids 33,82 Fecal matter, food habit studies with 28 Feed(s) additives used for flavor 138 ingredients 131 intake and production, voluntary .... 132 Feeding 93 activities of grackles 161 behavior on physiological systems, dependence of controller of 135 chemical senses and 104,136 experiences, maternal 28 responses of red-winged blackbirds 159,160 Felids 102 Feline attractant 99


170


68 Fennel 138 Fractionation procedure, urine 5,6,26,52,115 Fenugreek seed 138 Fructose 106 Fermentative scent sources 79,83 Fungiform papillae taste system cat 106 Fermented egg product as a coyote major chemical stimulus factors in attractant 86 carnivore 121 Ferret 88 neurophysiology of 104 Fiber content of the food 25 34 Fir seeds, Douglas 156 2-Furaldehyde Fixed-interval cells 56 Fixed-ratio (FR) cell 55 Flavor 3,6 chemistry 117 Galactose 70 of carnivore taste systems 102 Gas-liquid chromatography 26 components of whole grain 36 Genetic factors enhancers, protein 31 Geniculate ganglion chemoresponsive units of cat 107 with extracted or volatilized compounds, intensifying 33 Geniculate ganglion of the dog and cat 108 99 feeds, additives used to 138 Geraniol 22,79 imprinting, olfactory or food 27 Gerbils Ginger 138 preferences by individual experi ence, modification of 1 research, animal 1 3,5,6,14,22,23,26 Flavoring agents 31,33 Glucose homeostasis mechanism, blood 24 Flesh foods, cat taste neuron level of circulating 25 responses to compounds in 118 3 Floral 96 Glucoside Glucostatic theory 24 Flowering time of wild ginger, 2,3 slugs and 154 Glycine Glycyrrhizin ammoniated 138 Food(s) 8,130,136 available, criteria for making 59 Goats 153 aversion process, conditioned 11 Goldfish Grackles 158 cat taste neuron responses to feeding activities of 161 compounds in flesh 118 Grain for domestic pets, developing flavor components of whole 36 palatable 141 pigeon's choice of 47 factors influencing the acceptquality for the pigeon 62 ability of 45 flavor imprinting 12,27 rate on net energy available for growth, dependence of 134 habit studies with stomach content rations fed to milking cows 132 or fecal matter 28 Grasses, compounds and oil yield methodology of behavioral testing with selected 97 associated with development 30 in animal 43 Great Tit Growth, dependence of grain rate on odors as area attractants to net energy available for 134 rodents for control 31 Guinea pig 4,6,9,27,78,83 preference behavior in birds and 136 mammals 21 Gustation -producing animals, diets for 129 Gustatory chemical stimuli, species response to 93 selection and chemical senses 104,136 Gustatory sensitivity of vertebrates selection, preferences, experience, and some invertebrates 94 neophobia, and neophilia in .... 15 136 Fox(es) 36,102 Gymnemic acid red 27,85 anal sac secretions, aerobic H bacteria in 81 Hamster 78,79 bacterial action and chemical 47 signalling in 78 Head receptors 14 tissue extract 86 Hedonic factors Fractionation, chemical 67 Hedonic (pleasurable) taste s t i m u l i 2 2


171

INDEX

Hemp 62 Herbivore 4 Heterocyclic compounds 119 associated with meatflavorin humans 125 neural responses elicited by 114 Heterocyclic stimuli in the carnivore .. 125 Hexanoic acid 34 N-Hexylamine 34 D-Histidine 3 Hogs 22 Homeostasis mechanism, blood glucose24 Hormones, sexual 25 Human(s) 93 to amino acids, taste responses of .... 124 comparisons 122 heterocyclic compounds associated with meatflavorin 125 Humen, dried 3 Hungers, specific 1 Hyaenas 10 Hydrochloric acid 3,30 I Imidazole compounds 118 Imidazole ring 106 Indian mongoose 83,85 Indole 83,125 Ingestion, immediate consequences of 46 Ingestion, long term consequences of 47 Ingestional methods 44 Ingredient selection 144 Inorganic salts, monovalent 117 Inosinic acid 136 Insects 93 Instrumental method 52 Instrumental test apparatus 54,59 Insulin level 25 Intake of saccharin 51 Intake of sucrose 51 Interval cells,fixed-and variable56 Iodobenzene 136,137 Iridolactones 99 Isobutylamine 34 L-Isoleucine 108 Isovaleric acid 79 Isovaleric aldehyde 34 Invertebrates gustatory sensitivity of .. 94 J Jaguars Jojoba, seeds of

5 152 Κ

Ketones

99

L Laboratory procedures 53 Lactic acid 3 Lactose 3 Leopards 5,102 D-Leucine 3 L-Leucine 3 Linseed oil 34 Lion 80,82,83 Lithium chloride 2,3,48 -treated meat 157 Long term consequences of ingestion 47 Long term tests 49 M Magnesium chloride Magnesium sulfate

3 3

Maltose 3,6,52 Mammalian fermentative scent sources 84 Mammalian origin, microbially generated chemical signals of 78 Mammals 93 bacterial action and chemical signalling in 78 body surfaces of living 78 food preference behavior in 21 to naturalflavor,stimuli, responses of 6 physiological adaptiveness of wild .. 21 to synthetic tastants, responses of .... 9 Marketing, test 148 Matching law „ 61 Maternal feeding experiences 28 Maternal pheromone 28 Meat flavor in humans, heterocyclic compounds associated with 125 protein material 144 supplying animal protein require ments for the world population 130 Medicine effect 12 Menthol 94 Mestranol 158 Metabolic reflex, preparative 46 Methiocarb bird repellent 30,158,160 Method, instrumental 52 Methodology of behavioral testing associated with development in animal foods 43 Methyl esters of coyote estrus urine acids 76 6-Methyl-5-hepten-2-one 99 Mice 12,26,27,33,46,78


172


Microbially generated chemical signals of environmental and mammalian origin Micro-ecosystem, anal sac Microflora Microtine rodent population levels ....

Ο

78 N-Octylamine 80 Odor(s) as area attractants to rodents 80 for control, food 27 preference test Microtus montanus 95 primary Milk-supplying animal protein repellent effects requirements for the world sensation population 130 time spent at Millet, white 46 Odorants, attractancy to Mimicry model of selective prédation 30 Mink 80,83 Oil(s) of plants, essential Molasses 137 yield with selected grasses Momentary satiation 57 Monellin 3 Olfaction Mongolian gerbil 79 Olfactory chemical stimuli, species Mongoose, Indian 83,85 response to Monkey 85 imprinting squirrel 8 Monosodium glutamate 136,13 Monosodium-L-glutamate 3 Morphine 46 Opossum Mountain sheep 21 Oral chemoreception, sensory structures in Musk deer 99 Musky 96 Organic conditions Organic states, special Organics of plants, volatile Oxygen compounds Ν Oxygen factor Naringin 3 Neophilia in food selection 15 Neophobia 48 in food selection 15 Nepetalactone 99 Neural groups, chemoresponsive 106 groups, geniculate ganglion of the dog and cat chemosensory 108 responses elicited by heterocyclic compounds 114 Neuron responses to compounds in flesh foods, cat taste 118 Neurophysiology of fungiform papillae taste systems 104 Nicotine 3 Nitric acid 3 Nitriles 152 Nitrogen factor 121 Nitrogen heterocycle pK values Ill Nucleotides 108,118 Nucleotide factor 121 Nutrient requirements 129 Nutritional ecosystem, carnivores in 103 factors 23 guidelines 149 Nutritious diet 14 a

34 31 34 96 33 96 72 88 33 99 97 136 93 27 100 105 46,48 46 99 118 121

Ρ

P. miragilis

Palatability Pandas Peanut oil Peas, Canadian Pelargol Pepperminty Peptides Peripheral stimulation Pest birds Pest, vertebrate Pets, developing palatable foods for domestic Phenolics Phenylacetic acid D-Phenylalanine L-Phenylalanine Phenylethanol Phenylpropionic acid Pheromone maternal O-Phophoral ethanolamine Phylogeny Physiological adaptiveness of wild birds and mammals


80

149 102 34 46 99 96 118 47 30 150 141 152 79,83 3 3 99 83 99 28 112 15 21

INDEX

173

Physiological ( continued ) aspects of animal behavior 92 influences 24 systems, dependence of controller of feeding behavior on 135 variables 43 Phytic acid 108,112 Pigeons 26,30,43,46,56,58,61 grain quality for the 62 preferences of 47,59 Pigs 130,137,138 Plants chemical defenses of 151 essential oils or volatile organics of 99 substances, secondary 152 Poison avoidance by animals 155 Poisons 48 Polecats 36,80 prey-catching behavior of 2 Polyamines 11 Polydipsia 2 Porcupines 8,9 Potassium chloride 3 Potentiators 31 Prédation, mimicry model of selective 30 Preference(s) bait additives 32 behavior in bird and mammals, food 21 effect, rice varietal 28 in food selection 15 by individual experience, modification of flavor 11 of pigeons 47,59 ratio 58,61 responses of chickens, taste 29 ofricefieldrats for rice 35 test, odor 34 testing 149 Preparative metabolic reflex 46 Previous experience 48 Prey-catching behavior of polecats ... 27 Primary odors 96 Principle of least consummately effort 50 Product fabrication 145 Projection zones of cat geniculate ganglion chemoresponsive units .. 107 L-Proline 108 2-Proline 115 Pronghorn 79 N-Propylamine 34 N- ( η-Propyl ) benzylamine 34 Protein flavor enhancers 31 material, meat 144 source or concentrate, vegetable ... 144 supplement, ration-balancing 144

Protein (continued) requirements for the world population, meat and milksupplying animal sweet-sensitive

130 94

Psychobiologists Psychophysics, animal Pungent Putrid Pyridine compounds Pyrole

43 44 96 96 118 125

Proteur spp

Quail bobwhite Quelea Ouinine(s) compound

Q

80

30 156 21,31 14,22,94 3

R Rabbits Raccoons Radish, wild Raffinose Rat(s)

8,9 100 94 3 11,22,23,26,27,31, 33,43,50,56,85,155 instrumental testing apparatus for the 54 laboratory 6,9 Norway 25 pups 138 rice bait consumption of 29 ricefield 25,28,35 sucrose intakes in 47 sweet tooth of the 46 taste preferences 4,10 Ration-balancing protein supplement 144 Ratio(s) cell, (FR), fixed55 cell, (VR), variable55 preference 58,61 schedules 57 Receptor activation by a cat group, molecular circuitry associated with 123 Receptors, head 47 Red fox (see Fox, red) pepper 138 squill 48 Red-winged blackbird 30,155,158-160 Reinforcement ratio 61 schedules, concurrent 58 schedules, simple 55


174


Repellency, applications of conditioned Repellant(s) bird (methiocarb) effects, odor to protect crops from vertebrate pests Reproduction 5-Ribonucleotides Rice bait consumption of rat colonies preference of ricefield rats for varietal preference effect volatiles additive zinc phosphide-treated Rodents for control, food odors as area attractants to heteromyid population levels, microtine Rosetone Rubbing-rolling

Seed(s) (continued) mottled and grey 154 planting, corn 161 Senses, food selection and chemical 104,136 Sensory 150 information 14 93 structures in oral chemoreception .. 105 31 systems, chemical 104 Sexual hormones 25 29 Sheep 8,130,136 35 bulbectomized 137 28 Short term tests 49 36 Shrews 27 36 Signals, chemical input 120 22,93 Signals, microbially generated chemical 78 31 Signalling in mammals, bacterial 152 action and chemical 78 156 93 30 33

9 7

Simple reinforcement schedules 55 Skunk 80,93 Slug, native 153,154 S Smell 92,99 S. faecalis 80 Sniffing 71,136 Saccharin 3,26,46,48,50 Sodium 23 concentrations 54 chloride 2,3,6,30,115 intake of 51 fluoride 3 Salicylaldehyde 30 saccharin (sweet) 9 Salience of stimuli 14 Sorghum 31 Salt 94 Sour stimuli, responses of mammals Salts, inorganic 118 to natural 6 Salty stimuli, responses of mammals Sour substances 8 to natural 6 Soy sugar 32 Salty substances 8 Soybean oil 34 Sassafrass oil 34 Special organic states 46 Satiation, momentary 57 53 Scent marking 74 Species/response arrangements Species response to gustatory and Scent sources olfactory chemical stimuli 93 environmental 78,84 31 fermentative 79,83,84 Spiced diet 31,33 carrion 83 Spices, empirical screening of 134 mammalian 84 Steer, cost of growing a Stimulation, peripheral 47,48 Schedules 14 concurrent 60 Stimuli, salience of reinforcement 58 Stimulus factors in carnivore fungiform papillae taste systems, major 121 ratio 57 simple reinforcement 55 Stomach content, food habit studies with 28 Scraping 74 Streptococci 80 Screening offlavoringagents, 3 enhancers, and spices, empirical 31 Strychnine 25 Sebaceous tissue 80 Sublethal illness effects 3,36 Secondary plant substances 152 Succinic acid Sucrose 3,5,6,22,50,56 Seed(s) intake of 47,51 Douglas fir 156 octaacetate (bitter) 3,9,22,30 dove weed 152 2,32,94,137,144 jojoba 152 Sugar(s)


175

INDEX

Sweet to humans, compounds tasting sensitive protein sodium saccharin stimuli, responses of mammals to natural substances tooth of the rat Sweetness enhancers Synthetic taste compounds Τ

Tigers 3 Time spent at odor 94 Tissue extract, fox 9 Toxicological problems Trimethylamine 6 Triphosphate nucleotides 5 L-Tryptophan 46 Turtles, snapping 31 D-Tyrosine 9 L-Tyrosine

5,80 72 86 14 83,84 119 3,108 12 3 3

U

Tastants, responses of mammals to Urea 3 synthetic 9 Urine acids, methyl esters of coyote estrus 76 Taste 57,92 attractive to the coyote, chemical neuron responses to compounds in fractions from estrus 66 flesh foods 118 collection, estrus 66 preference fractions bioassay of 67 cat, rat, and cavy 10 chickens 2 rat, cat, and guinea pig 4 responses of carnivore and man to V amino acids 124 Valerone 34 stimuli, hedonic (pleasurable) or 94 aversive (repellent) 22 Vanillin 56 stimuli and responses 2 Variable-interval cells,fixedand Variable-ratio (VR) cell 55 systems Vegamine 32 cat and dog fungiform papillae 106,110 Vegetable protein source or concentrate 144 flavor chemistry of carnivore 102 Veltol plus 32 major chemical stimulus factors in carnivore fungiform papillae 121 Vertebrates, gustatory sensitivity of .. 94 Vitamin A deficiency 24 neurophysiology of fungiform papillae 104 Volatilized compounds, intensifying flavor with 33 thresholds as a function of body 9,27,33 state, altered 48 Voles Teeth, shearing (carnassials) 103 W Terpene hydrocarbons 3 Terpenoids 3,152 Water activity 142 Territorial markers ...93,99 Weasels 102 Test Wheat 62 area, coyote 68 White millet 46 durations 4 Wintergreen oil 34 marketing 148 Wolf 86,93 Tetramethylthiuramdisulfide (TMTD) 158 X Tetrasodium pyrophosphate 112 Theobromine 3 Xylose 3,29 Thiamin compounds 118,119 Thiamine (vitamin Bi) deficiency 23 Ζ Thiamine in chickens and rats, rice 36 appetite for 24 Zinc phosphide-treated Thaumatin 3 Zymino 32


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