MANAGEMENT OF WILDLIFE IN CAPTIVIT Y:
TAL ATHEROSCLEROSIS

IN GROUP AND SOCIALLY
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Thesis for the Dogreo of Ph. D.

MICHIGAN STATE UNIVERSITY
Prem Shankar Prasad
15965

LIBRARY

Michigan State
University

 

This is to certify that the

thesis entitled

Management of Wildlife in Captivity: Experimental
Atherosclerosis in Group and Socially Isolated
Japanese Quail (Coturrnix Coturnix Iaponica)

presented by

Prem Shankar Pras ad

has been accepted towards fulfillment
of the requirements for

Ph. D. degree in Fisheries & Wildlife

 

0-169

ABSTRACT
MANAGEMENT OF WILDLIFE IN CAPTIVITY: EXPERIMENTAL

ATHEROSCLEROSIS IN GROUP AND SOCIALLY ISOLATED
JAPANESE QUAIL (COTURNIX COTURNIX JAPONICA)

by Prem Shankar Prasad

Management of wildlife in captivity, from the standpoint
of managing a zoological garden, is reviewed. Administra-
tive organization of a zoological garden, with particular
reference to biology of animals, is discussed. Problems of
confinement, limited space, proper display of animals,
feeding, breeding, exercise, training, handling and disease
control are discussed in some detail. These are based upon
the recent advancements made in the field in the 420 zoolog-
ical gardens of the world. The many links between zoo
biology and animal psychology are given prominence through-
out the text.

Zoological gardens of today are not only a place for

+
public entertainment, they are also biological institutions
and should fit the community needs in size and service.
Both the people managing a zoological garden, and those
visiting a zoo, thus, have certain responsibilities. These
are discussed throughout this work.

Recent advancements in the field of animal nutrition

and animal sanitation, and their application in the

Prem Shankar Prasad

I

zoological gardens, have eliminated most of the infectious,
mal—nutritional, and deficiency diseases. However, arte-
riosclerosis, among "well-fed" and well-kept varied wildlife
of zoological gardens is posing a constant problem.

In an attempt to evaluate the effect of environmental
factors on dietary-induced atherosclerosis, Japanese quail
(Coturnix coturnix japonica) were held in groups and in
complete social isolation caged singly (each provided with
one square foot of space). The experimental birds were
kept on an atheromatous diet, while the diet for controls
was diluted with unsaturated vegetable oil to make the two
rations comparable.

A significant increase in the weight of adrenal glands
was found in all the birds kept either at higher density
level, or caged singly in complete social isolation. This
correlated well with the amount of social interaction in the
birds. Social hierarchy in the males develops after they
attain sexual maturity (at about 8 weeks).

Adrenals were heaviest in the birds kept singly in com-
plete social isolation. Olfactory, acoustic, or visual
stimulation seems to evoke adrenal responses in these birds.

A highly unnatural situation in the form of intense

crowding, or complete social isolation, enhanced atherogenesis.

Prem Shankar Prasad

Isolation of various interacting factors, and the process
or processes involved regarding their neural mechanism, how-
ever, is not yet clear.

A survey was made of the electrophoretic pattern of the
serum protein of birds kept in the natural and experimental
conditions. Each pattern from the experimental group was suf-
ficiently unique to differentiate from the control birds.

Fundamental differences in the protein components of
normal and experimental birds are described. These were
generally in the form of increased beta-globulins and decrease
globulins. It has been

in the components of alpha and alpha

1 2

suggested that electrophoretic pattern of serum protein may
even be a reliable guide of the developmental stage of the

disease.

MANAGEMENT OF WILDLIFE IN CAPTIVITY: EXPERIMENTAL
ATHEROSCLEROSIS IN GROUP AND SOCIALLY ISOLATED
JAPANESE QUAIL (COTURNIX COTURNIX JAPONICA)

BY

Prem Shankar Prasad

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Department of Fisheries and Wildlife

1965

337.21. 9

ACKNOWLEDGEMENTS

The author wishes to express his sincere gratitude and
deep appreciation to Dr. P. I. Tack, Professor and Head of
the Department of Fisheries and Wildlife, for his guidance,
advice during the entire course of the investigation and the
preparation of the manuscript. Without the financial support
of the Department, undertaking of this investigation would
not have been possible.

The author is indebted to Dr. Rollin H. Baker, Profes-
sor of Zoology; Dr. Miles D. Pirnie of the Fisheries and
Wildlife Department; and Professor Louis Twardzik of the
Department of Resource Development; all of whom gave so
generously their valuable time for the critical review of
the manuscript and the helpful suggestions.

For invaluable assistance by providing birds for this
investigation, the author is deeply indebted to Dr. Robert
K. Ringer, Department of Poultry Science, and to Mr. Ralph
A. Ernst, a doctoral student in the same department. Dis-
cussions with Dr. Ringer, and his various suggestions, were

equally informative and helpful. Special thanks are due to

ii

Dr. Howard C. Zindel, Head of the Poultry Department, for
providing pen, and the space to house the experimental
birds.

It is with deep and sincere appreciation that the
author expresses his gratitude to Dr. John A. King, Pro-
fessor of Zoology, for the appointment to a technical
assistant and thus providing financial support to the
author which enabled him to pursue his studies in the
United States. Thanks are also due to the Government of
Bihar, Patna, India, for partial support of this study.

Lastly, the author is grateful to his parents for
their constant encouragement and moral support without

which this project would have remained incomplete.

iii

TABLE OF CONTENTS

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . 1
PART I
MANAGEMENT OF WILDLIFE IN CAPTIVITY

ADMINISTRATIVE PROBLEMS OF A ZOOLOGICAL GARDEN . . . . 5
Organization of a zoological garden 5
History of zoological gardens 6
Governing authority 11
Administration of a zoological garden 13
Functional division of a zoological garden 14
Curator 15
Animal keeper or the animal attendant l9

OPERATIONAL PROBLEMS OF A ZOOLOGICAL GARDEN . . . . . 24
Problem of confined space and its biological

significance 24
Housing and display of animals 27
Problems of food and feeding 40
‘ Quality and quantity of food 43
Feeding the herbivores 43
Feeding the carnivores 45
Feeding the piscivores 47
Feeding the omnivores 48
Social and psychological problems in feeding 52
Exercise and training 55
Disease control and animal restraint 62
Role of veterinary services in a zoological
garden 65
Problems of disease and disease prevention 65
Problems of animal restraint 72
Interaction of public and zoo tenants 75
PART II

EXPERIMENTAL ATHEROSCLEROSIS IN GROUP AND
SOCIALLY ISOLATED JAPANESE QUAIL
(COTURNIX COTURNIX JAPONICA)

INTRODUCTION . . . . . . . . . . . . . . . . . . 81
MATERIALS AND METHODS . . . . . . . . . . . . . . . . 87

iv

Acquisition and handling of chicks

Housing
Management

Handling of birds

Collection of materials
Electrophoretic methods

Mounting the membrane on the bridge

Sample application

Staining, cleaning, and mounting the strips

in the plastic envelopes
Evaluation of the separated serum protein

RESULTS . . . .

DISCUSSION . . .

Body weight and adrenal weight
Electrophoretic pattern of serum proteins

SUMMARY . . . .

LITERATURE CITED

Body weight and adrenal weight
Electrophoretic pattern of the serum protein
Normal birds
Birds kept on cholesterol-mix diet
Birds kept on non-cholesterol diet
Incidence of disease in the experimental
control birds
Behavioral differences

and

Page

87
87
88
90
90
92
94
95

96
97

99
100
102
102
103
109

112
113

119
119
121
126

129

LIST OF TABLES

Page
Japanese Quail Breeder Ration . . . . . . . 89
Body Weight and Adrenal Weight Data for
Isolated and Group Raised Japanese Quail . . 101
Summary of Electrophoretic Pattern of Serum
Protein of Normal Male and Female Japanese
Quail O O 0 O O O O O O O O O O O O O O O 0 108
Summary of Electrophoretic Pattern of Serum
Protein of Japanese Quail Kept on
Cholesterol Diet . . . . . . . . . . . . . . 110
Summary of Electrophoretic Pattern of Serum
Protein of Japanese Quail Kept on Non—
cholesterol Diet . . . . . . . . . . . . . . lll

vi

LIST OF FIGURES

 

Page
Analytrol showing a scanned graph . . . . . 104
Electrophoretic pattern of the serum of a
normal female Coturnix quail (Coturnix
coturnix japoniga) . . . . . . . . . . . . 106
Electrophoretic pattern of the serum of a
normal male Coturnix quail (Coturnix
coturnix japonica) . . . . . . . . . . . . 106
Formation of atherosclerotic plaque in the
lumen Of aorta O O O O O O O O O O O O O O 114
Formation of atherosclerotic plaque in the
lumen of aorta . . . . . . .‘. . . . . . . 114
An advanced case of atheromatous plaque in
the aorta . . . . . . . . . . . . . . . . . 116

vii

INTRODUCTION

A comprehensive literature exists on how to manage
domestic animals, dealing at length with every conceivable
detail. Feeding, breeding, transmission of hereditary
characters, pedigree, distribution, pathology, training
and so on have long been the subject of profound research,
and each has become a specialized branch of an impressive
science. On the other hand, the knowledge of how to keep
wild animals in captivity can hardly boast of even the
most general outline; all it has to show is a collection
of more or less fragmentary pieces of advice and some facts.

Despite such limitations, the zoological gardens of
today have progressed far beyond the scope and status of
a mere menagerie. During the present century, great strides
have been made toward the development of management tech-
niques that can satisfy the physical as well as psycho-
logical needs of the animals and at the same time allow
them to be shown so that at least some segments of natural
habitats and life cycles are illustrated. Changing world

conditions that endanger the wildlife of many areas, have

brought the zoological gardens into new prominence in the
field of propagation of threatened species. Also, zoos
have far reaching values in the field of education and
research.

The biology of zoological gardens, however, is such a
complex field that it is difficult for a single individual
to get a complete view. It ranges, for example, from zo-
ology to psychology (including human psychology), and from
ecology to pathology. The present study, therefore, en-
deavors to provide a review of some useful relationships
between the most important of these aspects.

The first part of the text deals with some major prob-
lems of a zoo which provide the basis for the present study.
The contents in this section are discussed under two
separate headings:

a) Administrative problems of a zoo

b) Operational problems of a zoo.

These are further subdivided into smaller groups and dis-
cussed in some detail.

The second part presents an independent research deal-
ing with an emerging problem of the pathological aspect of

captive animals, the so-called "disease of civilization."

It presents an intricate relationship between the physio-
logical and psychological state of the animal, a clear

understanding of which is still far from accomplished.

PART I

MANAGEMENT OF WILDLIFE
IN CAPTIVITY

ADMINISTRATIVE PROBLEMS OF A
ZOOLOGICAL GARDEN

The major consideration under this heading is the
administrative organization of a zoological garden. Through
administrative planning, long and short range objectives are
determined, policies are formulated, organizational structure
is streamlined for efficiency, and the work of the organiza-
tion is given coordination, direction, and control. An or-

ganization, thus, accomplishes its objectives through people.

Organization of a zgglogicalpgarden

Inconsistency in the organization of zoological gardens
is such that scarcely two are alike. Most zoological gardens
of the world seem to have developed quite independently from
one another--a fact which seems fundamentally wrong with such
an important organization. A number of factors may have con-
tributed in varying degrees to this independent growth. Poor
communication, in some cases, undoubtedly resulted from the
great distances separating some zoological gardens. A
friendly spirit of competition, as it does to a certain ex-
tent today, may have existed, creating a delaying effect on

the flow of new ideas.

Hediger (1950) in his book "Wild Animals in Captivity"
stressed that
Zoological gardens cannot have isolated existence today.
They are necessarily linked up with the long history of
development which the keeping of wild animals has under-
gone in the course of centuries, and this means they
have responsibilities. Every zoological garden is a part
of the whole system and must fit into the picture along
with all other zoos. The important knowledge gained in
past and present zoological gardens demands suitable
recognition.
To better understand the organization of a zoological
garden it seems imperative to mention a brief historical

badkground of zoological gardens from the beginning up to the

present day.

History of zoological gardens

No record is available to indicate who founded the first
zoo. Man's enthusiasm for keeping pets probably was the
original impetus; a desire to gain favor by presenting some
strange beast to a ruling potentate was probably another.
Certainly the custom of collecting wild animals is ancient.

The earliest zoological garden for which there is a
definite record was established in China by the first Emperor
of the Chou Dynasty, who reigned about 1000 B.C. (Cadwalader,
1949). This was known as the "Intelligence Park," and its

name implies that it served an educational purpose.

The keeping of animals for pleasure and exhibiting them
for show or entertainment was highly regarded by the Egyptians,
the Greeks, and the Romans (Berridge, 1932).

The great animal collection of Ptolemy II (309-246 B.C.),
when exhibited in a procession through the city of Alexandria,
as a part of the festival of Dionysus, took all day. A re-
markable assemblage of animals formed a part of this parade.
Elephants and groups of other animals in harness drawing
chariots, camels laden with spices, sheep, oxen, bears, leop-
ards, lions, and birds in cages were included. 24,000 hounds
also passed in review, and there were 150 men carrying "trees
to Which were attached wild animals and birds of all sorts"
(Jennison, 1937).

Hediger (1950), speaking about the evolutionary history
of keeping wild animals by man from paleolithic time to the
beginning of the twentieth century, divided the subject into
three main groups:

1. The age of cults: The incentive for keeping animals
was based on religious beliefs, especially for use as sacri-
fices. In ancient Egypt, animals such as lions, baboons,
crocodiles etc. were considered sacred and suffered from a
special type of pampering. These "animal gods" were often

kept in temples and surrounded with pomp fatal to them. They

were provided with food chosen on the basis of what would suit
human tastes; were bathed, anointed and perfumed. The sacred
crocodiles of Lake Moeri that came out at the call of their
priest keepers were decked out with bangles and necklaces
(Loisel, 1912). These unnatural treatments caused patholog-
ical conditions in many, as Lortet and Gaillard (cited by
Loisel, 22, git,) have pointed out in their exhaustive re-
searches on mummified animals.

2. The profane age: The chief consideration being

a) usefulness;
b) entertainment (animal fights and hunting).
3. The scientific age: The interests being mainly
a) systematics and anatomical;
b) ecological and psychological.
These divisions show how the emphasis in man's attitude to
towards animals gradually shifted and reoriented.

As civilization spread westward and northward, the custom
of keeping wild animals continued. During the middle ages they
were held captive mostly for sports. Eventually, zoological
gardens were established in many of the larger cities of cen-
tral Europe, France, and in England. The collections in Paris
under Louis XIII and Louis XIV were well known.

The zoological gardens now existing in the world are all

derived in some manner from Jardin des Plantes of Paris which
appeared at the end of January, 1793 (Loisel, 1907). "This
was a place set aside for the study of nature, in the inter-
ests of science and the liberal arts, for the scientists and
the artists (Loisel, 22, 222,).

Of all the zoological gardens of the world, the Zoolog-
ical Garden of London seems to be the oldest and the largest
(both in total number of specimens and number of species
exhibited; total specimens 5,654; number of species on ex-
hibit 1,544). It was founded by the Zoological Society of
London in 1826 for the purpose of "the advancement of zoology
and for the introduction into England of new and curious ani-
mals" (Loisel, 22, 222,). It was incorporated by Royal Charter
in 1829. This garden belongs to the Zoological Society of
London which serves the dual purposes of maintaining the
garden as well as publishing Proceedings, Symposia, Trans-
actions, Nomenclator Zoologicus, Zoological Records, Inter-
national Zoo Yearbook, Zoo Magazine, Zoo Guide, Zoo Plan and
various scientific and non-scientific leaflets.

On North American continent, the Zoological Garden of
Philadelphia is the first and foremost zoo, equipped with a
modern research laboratory. It is affiliated with the Uni-

versity of Pennsylvania. The garden, likewise, is managed

10

by the Zoological Society of Philadelphia and is governed by
a Board of Directors, none of whom receive remuneration.

Philadelphia Zoological Garden was founded by the Zoo-
logical Society of Philadelphia in 1859 and initially covered
an area of about 33 acres. Currently the garden occupies over
42 acres of land and is exhibiting 1,595 specimens represent-
ing 555 different species of mammal, bird, reptile, and
amphibian. It publishes a quarterly magazine and zoo guide
besides several scientific papers coming out of its Compara—
tive Pathological Laboratory.

Jarvis and Morris (1961), emphasizing the need of a
zoological garden in the field of propagation of threatened
species, have said, "The policy of zoos all over the world
must surely be conservation; whether rare species of animals
are preserved in a natural or captive environment is of sec-
ondary importance." India, however, where some of the noblest
and most beautiful animals of the world are to be found, is
most backward in this respect. In his forwording note for
"The Wild Life of India" by E. P. Gee (1964), the late Prime
Minister Jawaharlal Nehru has said, ". . . in no country is
life valued in theory so much as in India, and many people
would even hesitate to destroy the meanest of the most

harmful animals. But in practice we ignore the animal

11

world." Since the inception of the Indian Board for Wild
Life in 1952, a list of 13 rare fauna of India was published
(Gee, 1964). At least two of them, the pygmy hog and the
Indian Cheetah, are now believed to be extinct. The Indian
Lion (Panthera l§2_2ersica), whose existence in their natural
habitat is most threatened, however, is doing well at the
Calcutta Zoological Garden (census of rare mammals in cap-
tivity, International Zoo Yearbook, 1961).

Currently, in India, there are 10 zoological gardens of
which the Victoria Zoological Garden, Bombay, is the oldest
and the Alipore Zoological Garden, Calcutta, is the largest.
The Alipore Zoological Garden was founded in 1875 by the
Government of Bengal. It now occupies 50 acres of land and
has a total exhibit of 1,965 animals. The financial support
for the garden is met by the State Government of West Bengal
and a part of it comes from the city corporation. The Zoo
has an attached laboratory for pathological and zoological
research and also publishes an administrative report which is
distributed free of cost. Display and housing for most of

the animals, however, are not very up-to-date.

Governing authority

 

Individual zoological gardens can operate under a

variety of governing authorities. One means of control may

12

be successful in one community but inadequate in another and
local conditions generally determine what type of governing
responsibility will be established.

The superintendent or the director of a zoological gar-
den may have authority vested in him through the city manager
or the city commissioner or the heads of such departments as
public works or animal husbandry. Under any system, however,
the cooperation of a good zoological society or an advisory
board of influential and interested citizens can be of great
value in promoting the growth of the prOgram and maintenance
of high garden standards.

A zoological society is a group of interested and often
influential people who may direct or act as an advisory body
in the development and operation of the garden. Members of
the society are more likely to take greater interest in the
garden.

The primary objective of the society's interest is to
place the development and operation of the institution in the
control of a non-profit organization, free from political
influences and unaffected by frequent changes in the admin-
istrative affairs of the zoo (Mitchell, 1935). For the zoo-
logical garden it provides continuity of management, and to

the officials and employees permanent employment during

13

faithful and satisfactory service, both vital features in the
building up of any organization. For other advantages of a

zoological society to a zoological garden see Luehrmann (1931L

Administration of a zoological garden

The director or the superintendent is in charge of over-
all operation of the garden.‘ He directs and carries out the
policy and is directly responsible to the Board of Directors.
Divisional supervisors are directly responsible to him. It
is the director's duty to plan, coordinate, and direct the
work of the entire zoological garden. He is assisted by his
secretarial section in matters incident to the shipment and
receipt of specimens, including insurance and custom clear-
ance, also mail receipt and transmission, public relations and
interpretive programs, and maintenance of the central files.

In most modern zoos, a separate public relations divi-
sion integrates publicity and other informational activities
through all kinds of public communication media. It deals
with membership fees and subscriptions, signs and posters,
publications, public information, radio and TV programs, lec-
ture series and reference library, and guided educational as
well as public tour services.

The public service division operates restaurants and

refreshment stands, vending operations, films and other

l4

accessories, and souvenirs of the garden. It also caters to
special groups and the general public through picnic facilities.
In almost all zoological gardens there is a separate
security division which may be further divided into building
service section and a patrol section. The duties are to
patrol and protect the garden properties, enforce security
regulations governing exhibits and visitors' behavior, super—
vise public comfort, direct visitors, and render first aid

service when and where needed.

Functional division of a zoologigaiigarden

Most of the big zoological gardens have their zoolog-
ical department divided into four separate units that include:

1. The animal clinic and the laboratory: The work of
this division is governed by a research director who super-
vises the veterinary work and other zoological research
projects. The veterinarian and a research team work directly
under him.

2. Ornithology division

3. Herpetology division which takes care of the
aquarium and the reptile house.

4. Mammalogy division.

The last three divisions are often the responsibility of a

15

separate curator who is further assisted by a team of workers
including a head keeper, a junior keeper, and several animal

attendants of various grades.

Curator

The curator of a division is subject to administrative
direction from the superintendent or the managing director of
the garden. He is responsible for the acquisition, health
and maintenance of the animals under his division. The di-
rector and the curator of a division work together, planning
and organizing long-range objectives of their respective
units.

The curator supervises the work of the personnel employed
in his division. He is responsible for feeding, breeding,
receipt and shipment of animals. The animal's environment
and its general health should be checked daily and any devi-
ation from normal reported to the veterinarian and to the
director. It is the responsibility of the curator to see that
the advice of the veterinarian is strictly followed. As a
zoological expert, the advice of the curator is important to
the efficient care, handling, and exhibition of the animals.

Another aspect of biological nature directly concerned
with administration is the complete inventory of all the

animals at hand. This, however, varies widely from one zoo

16

to another. It should give continuous information about a
number of similar species, as well as about many completely
different ones. Also, this should not be confined to the
individual, but should include the ancestors, the progeny,
and special events in life such as growth, accident, ill-
ness, breeding cycle, as well as special behavior, change
in social organization etc.

If possible, and if it does not involve too great ex-
pense, all animals must be weighed periodically and records
kept. This provides a valuable reference about growth and
development and also their general well-being. In almost
all zoological gardens, a separate card is maintained of
the periodic parasitic check of feces so that the parasitic
state of the animal may be compared at a glance. This
problem has efficiently been discussed by Peters (1933).

The capture of every animal means exclusion from its
natural environment and man incurs full responsibility for
providing adequate surroundings. The zoo, in this respect,
should not play its role only as a "living museum" or a
repository of the world's fauna. The surroundings should
be designed and maintained in such a way that the wild
animals housed in them not only live long in captivity but

also live in conditions where they can reproduce themselves.

17

The loss due to capture, thus, can directly be compensated
by releasing the offspring, in return for the individual
loaned, as it were, by nature. This simple remedy, however,
could be applied only in rare circumstances. One of the
best examples of this is the restocking of the Swiss Alps
with ibex from animals born in captivity. Hediger (22, 222,)
is correct in saying that there is
. only one criterion for suitable biological condi-

tions and that is success in breeding. When breeding

does not occur, something is wrong with the methods of

keeping the animals; if breeding does occur, it is

guarantee that the conditions are essentially right.

This important aspect should, therefore, be the great-
est challenge of the curator or the director of a zoological
garden, and every effort should be made to achieve this
goal. There are still many species that have not been
induced to breed in captivity, and our knowledge about their
physiology and ecology is seriously lacking. In the inter-
ests of the maximum breeding potential of the stock of
animals in all the zoological gardens of the world, close
cooperation on an international scale is desirable, quite
apart from its significance in many other fields.

Among zoo animals, a vast array of behavior of the

mother-child relationship may often be observed. Since

many kinds of teratological conditions arise due to

18

possible physiological disturbances, in the same way the
danger of qualitative or quantitative departures from the
normal may occur in every single element of behavior in the
mother-child relationship after birth. These deviations
from the normal, as often credited, are not always due to
under-nutrition. In some cases, close connection with hor-
mone metabolism has been shown. Interesting cases of in-
credible nature have been described by Yerkes (1929) among
his anthropoid apes. The mother may sometimes blow into
the mouth of newborn which are unable to breathe. Once a
mother chimpanzee, whose baby was not breathing, pulled its
tongue out with her lips and blew until it started to
breathe.

The hypertrophy of the maternal instinct, or the over-
exaggerated care of the young, however, may sometimes prove
fatal to the offspring, especially in the predatory animals.
In order to hide their young in a more secure place, they
drag them around by the scruff until they eventually suc-
cumb to exhaustion or even to serious injuries. Even a loud,
disturbing noise nearby might bring evidence of that odd
quirk of animal psychology which prompts carnivore mothers
in captivity to kill their young at the first suggestion of

danger (Brown, 1932).

l9

Expectant mothers should, therefore, be given special
care and individual attention by the curator. Breeding and
successful rearing of rare fauna of the world is one of the
zoo worker's greatest challenge, and its accomplishment is

the greatest reward.

Animal keeper or the animal attendant

An animal keeper or the animal attendant is directly
responsible to both the curator and the head keeper. The
general condition of enclosures and animals under his care
are reported daily to the head keeper. He must frequently
watch the behavior and condition of animals under his
charge for signs of illness, injury, or breeding condition.
He is often called upon to assist in crating, moving, and
shipment of animals.

Animals must be fed and watered strictly according to
the schedule and the keeper incurs full responsibility for
mishaps occurring due to any irregularity. In small zoos,
it is also the duty of the keeper to prepare and mix the
food of animals under his charge. Food containers and
drinking bowls must be cleaned and sterilized daily.

It is the duty of the animal keeper to keep the animal

quarters and their adjacent areas scrupulously clean,

20

especially during the visiting hours. It is extremely
important for the keeper to know the proper disinfectants,
vermin repellants, and other cleansing agents for different
situations since some may be dangerous to certain groups of
animals. Most zoos, however, are not in favor of using
disinfectants for cleaning the animal quarters (personal
communication, Freeman Shelly, Director, Philadelphia
Zoological Garden). Carnivorous animals of the cat family,
for example, are highly susceptible to phenol poisoning.

It is generally known among keepers of zoological
gardens that the same animal may react quite differently,
even in opposite ways, to two or more men of apparently
identical characters. Brown (1932) gives an account of a
one-man elephant at the Philadelphia Zoo which always dis-
approved of the keepers except her own regular one. A fine
specimen of an ibex, likewise, took dislike to a new assist-
ant keeper at Basle Zoo (Hediger, 1950).

Hediger (22, 222,), analyzing the situation on the part
of the keeper said that a man from the very start may
either be sympathetic or antipathetic to the animal.
According to him, in the best interests of both the man and
the animal, and for a better relationship between the

keeper and the charge, the pathetic attitude must be as

21

positive as possible on the keeper's side. It makes for
avoidance of accidents, and simplifies running of an
establishment.

Also, in addition to this pathetic and antipathetic
attitude between man and animal, there are certain people
who feel themselves unusually happy with wild or ordinary
animals, while others meet the situation with consistent
difficulties. Hediger further feels this character to be
innate and one that cannot always be acquired with the
right sort of training.

Katz (1937) states that animal behavior of the above
mentioned kind and their way of reacting is of a more
primitive nature and does not seem to represent the indi—
vidual emotional experiences which the animal transfers
from one category of people to another. He goes so far as
to say that animal behavior of this kind may even provide
a new basis for the typology of man. From the biological
point of view, therefore, the keeper should be selected
from men having sympathetic attitude and an innate inclina-
tion for animals. Wild animals are unusually sensitive
creatures, and their happineSs depends to a great extent on
the good qualities of the keeper.

Another important aspect of animal psychology having a

22

direct bearing on the management of wild animals in cap-
tivity is the assimilation tendency. The animal sometimes
considers a man as one of its kind, and treats him as a
member of the same species. It is sometimes difficult to
differentiate this assimilation tendency with that of
Lorenz's "imprinting" which was originally ascribed only to
birds (Lorenz, 1935). In contrast to imprinting, assimi-
lation occurs slowly in mammals, especially with older
animals (Hediger, 1950). This animalizing tendency of the
animal consequently incorporates man as a fellow member
into the social organization which has both practical
advantages and disadvantages. Assuming man occupies the
d-position (premier position), the advantages of being
accepted into their social organization are of several
kinds. The man enjoys the full privileges that the a-
animal in the group possesses, namely complete freedom of
movement, and all inferior animals must obey his orders
without question. Kohler (1931) reports that illness makes
a drOp down on the social scale, and so an especially
strong attachment to man or the keeper is often noticed in
sick animals, which is very useful for giving treatment.
The chief disadvantage of this animalizing tendency is

that the man thus accepted by the animal is often involved

23

in disputes over social precedence and is, therefore, bound
to assume the premier position unless he is willing to give
up permanently close contacts with the animal. It is for-
tunate for the man, however, that quarrels about social
rank among higher animals do not always end with physical
superiority. Another most unpleasant situation commonly
observed in zoos as a result of the animal's assimilation
tendency is during breeding season. Man thus favored by the
animal is also considered as his sexual rival and they
either try to fight over him or to mate with him. Roebucks
always regard man as a rival of the same sex and would

never miss a chance to attack him during breeding season.

OPERATIONAL PROBLEMS OF A ZOOLOGICAL GARDEN

Maintenance of native and.exotic wild fauna in captiv-
ity requires a wide knowledge of various aspects of their
biology and involves certain responsibilities, too. The
knowledge so far gained in this respect is limited and
scattered and one, therefore, will always find some gaps
and frequent omissions. It is only by the close cooperation
of all the biological research establishments of various
zoos throughout the world that this imperfect outline could
be perfected and improved. The subject matter under this
heading is divided into five different categories and each
discussed separately in some detail.

Problem 9; confined space and its
biological significance

Confinement of space for life in captivity is one of
the immediate and most controversial concerns of zoo hus-
bandry men. The popular conception of "golden freedom" is
purely a product of human imagination and is certainly not
a biological fact. Neither the fish in its shoal (Breder,
1959), nor the monkey in its troop (Chance and Mead, 1953),
is free as an individual. Carpenter (1942) significantly

remarks that the monkey or ape in its natural group in the

l l l. i I

25

tropical forests has its freedom of movement strictly
limited by the structure of its group.

Hediger (1950) speaking about this anthropomorphism has
said, ". . . how wrong, from many points of View, are
anthropomorphic methods of observation in biology. It is
also necessary to get rid of a lot of the anthropomorphic
tendencies still current in the practical management of wild
animals, and in the biology of zoological gardens." Ana-
lyzing the problems of confined space (bars, cages, fenc-
ings, and other means of confining animals) from the bio-
logical point of view, he said that to the animals its main
significance may be one of the following:

1. Prevention of flight reaction: The animal attempts
to get away from man made barriers not to liberty, or home,
but away from its chief enemy--man, and his surroundings
Which are perhaps biologically unsuitable. There are
several examples Where, once released or escaped from their
confinements, animals have voluntarily returned to their
cages. The animal does not cease to regard man as an
enemy immediately after capture. This decisive change of
significance occurs only in course of getting used to the
new situation or of becoming tame (Hediger, 1935).

2. Prevention of encounters--with animals of its own

kind and/or others.

26

3. Limitation of territory: This ideal situation
quite often is achieved in most zoological gardens. The
space given to the animal is defended against new arrivals
in the same way as they would do in nature. For this rea-
son, it is quite risky to put fresh arrivals with animals
that have already settled down. It is much easier to get
strange animals accustomed to each other by bringing them
together in a 222 home instead of introducing one to another
already settled in the quarters.

4. Protection against encounters: And finally, a cage
may mean to the animal a protection against unwanted con-
tacts with animals of the same or different species. Sheak
(1924) describes a very delightful observation made on
Sally, a tame chimpanzee. Sitting in a chair one morning
When a leopard roared ". . . the chimpanzee jumped to the
floor, ran to the cage, lifted up the door, hurried in, and
closed the door behind her. She recognized the fact that
the cage was as good a contrivance to keep enemies out as
to keep her in."

For display purposes, wild trapped animals are always
inferior to zoo bred specimens. Having known "freedom," it
takes.longer to get used to the new situation, sometimes

only at the cost of serious losses. Each animal lives in

27

its own specific world. The environment offers, as it were,
a reservoir of stimuli from which the subject constructs its
own specific world. By capturing it we utterly destroy the
animal's previous world, and put it into a different situa-
tion. The animal must construct an entirely new world.

This means an enormous task on the part of the animal, one
which is not easy for every animal to tackle successfully.
The transitional phase, from freedom to captivity, has been

discussed in detail by Hediger (1950).

Housing and qgsplay22§:animals

Broadly speaking, there are two possibilities in which
the animals in a zoological garden could be housed and
displayed:

1. Adjustment of the animal's locomotor capacity to
the given space;

2. Adjustment of the space according to the animal's
locomotor capacity.

The first category consists of such mechanical devices
as hobbles, nose-rings, collar chains, and ropes by which
the animal's locomotor capacity can be accommodated to a
confined space. Such devices, although usually applied
solely to domestic animals, may be suitable for certain

groups of zoo animals such as camels, elephants,and ponies.

(I‘ll

i ll. 1 i l l l (IL Ill Ill. li
l l

28

Furthermore, in birds, the primaries of one side of the
wing are clipped to cause asymmetry and thus restrict their
flight movements. It causes the bird to fly in a circular
course, or to drop back to earth, and they cannot escape.
In the Calcutta Zoo, this was the usual practice for most
birds of the family Anatidae kept in open enclosures. Since
the clipped feathers grow again the following molt, the
process must be repeated at least once a year for which the
bird must be caught again, with all the accompanying dis-
turbances and the risk of injury involved. To alleviate
this probable danger, there is an operation called pinion-
ing often performed on one of the wings of the birds.

The operation, as described by Young (1948), consists
of exposing the metacarpal of the hand by making the inci-
sion at right angles across the wing just external to the
outer wing joint. The metacarpal bones, excepting the
thumb, well above the joint, are then severed together with
the attached muscles and the wound is closed by appropriate'
sutures. This causes the wound to heal rapidly, giving a
smooth appearance on the end, nicely covered with feathers,
and leaves a presentable bird for exhibition. The birds,
however, should be kept quiet after the operation or

profuse haemorrhage, sometimes with fatal results, may

29

happen (personal communication, Dr. M. D. Pirnie, Mich.
State Univ.). It is therefore advisable to do this opera-
tion when the birds are quite young.

There is no indication of post-operative complications,
however, Lorenz (1940) reported a striking change in be-
havior in one grey goose. After the operation, the bird
showed symptoms of severe depression with marked loss of
appetite and went so much "off condition" that breeding
activity was almost nil the following spring. This, how-
ever, may be an isolated case due to individual idiosyn-
crasy and may not justify a sweeping generalization.

The only disadvantage of this operation is that the
birds are mutilated, even though slightly. To overcome
this difficulty, several workers have suggested an alter-
native called radial-neurectomy or tenotomy (Young, 22,
222,; Sperling and Faber, 1951). Under a suitable anaes-
thetic, a small piece of the radial nerve supplying this
’“appendage is removed. The bird's skeleton remains com-
pletely intact, and so does its normal appearance. The
operation simply prevents the bird from stretching out its
wing to its full length causing asymmetry as a result of
which the bird cannot fly. In powerful fliers (pheasants

and gulls), however, this operation does not seem to help

very much.

30

According to Hediger (1950), not all zoo birds are
pinioned. Among the medium and larger kinds, it is usually
the Anatidae (geese and ducks), the flamingos, the waders
and the web-footed birds, and less often the Phasianidae
are treated. Because of their marked sedentary nature, it
is often simple to induce the Phasianidae and the peacocks
to settle down in a particular place. This is also true
with domestic pigeons because of their marked homing instinct.

Means of confinement under the second category (page 27)
are aimed at adjusting the space according to individual's
motor capability and not the other way around. These may
further be grouped into:

a) Psychological or imaginary--true only for circus
animals and not practical in zoos. The animal, by special
training, learns to respect the ring or other such barri-
cades which present a psychological barrier to the animal.

b) Natura1—-such as pits, moats, ditches and land-
water barriers. These are called natural because similar
obstacles are usually met with in nature.

c) Semi-natural--such as fencings, bars, wire nettings.
These represent a close network of branches and dense
creepers of the wild which the animal usually avoids because

of possible injury.

31

d) Artificial-~such as glass, light, and electricity.
These are not encountered in nature as such.

The pit is the most primitive way of confining animals,
both literally and historically. Palaeolithic man made use
of this, at least temporarily, as a means of confining wild
animals prior to killing for food. There is evidence of
keeping bears, and even large mammoths, in hollows of the
ground from where the animals could neither jump nor climb.
Pits present a vertical barrier in contrast to ditches which
are horizontal. There are still some older zoos in differ-
ent parts of the world where such enclosures are used
especially for such large carnivores as bears. The Zoo-
logical Garden of Lucknow, India, still has some large
reptiles on display in straight-walled pits.

Moats differ from pits and ditches in the fact that
animals do not live at the bottom. Moats surround modern,
free enclosures on the level ground, the area of which
depends upon the locomotor capacity of the animal. These
moats may be dry or filled with water, when they serve
as bathing pools.

The greatest difficulty encountered in this respect is
that every species of animal has its specific type of

locomotion and each has its own maximum capacity for

32

overcoming obstacles in both horizontal and vertical direc-
tions. In the construction of such "open cages," therefore,
it is important to know the maximum locomotive capacity of
the animal concerned. Unfortunately, accurate judgment or
calculation of this critical maximum capacity for each
species is not easy, and underestimation has led to a series
of deplorable accidents (Mottershead, 1960). Fundamentally,
the only sound method is to work out the capacity for over-
coming the obstacles for as many individuals of a species
as possible, using observations taken from the wild as an
additional check. Currently, the London Zoological Garden
is experimenting with over 20 different species of mammal
to determine their maximum jumping distances (Morris, 1962).

Hediger (1950) has further warned that the capacity of
an animal to clear an obstacle in a normal, balanced state
of mind is far less than that which happens during extreme
conditions of stress and excitement. This excitement fac-
tor, therefore, should always be considered while planning
an open enclosure.

Bigalke (1961) recommended dry ditches or moats for
confining wild animals because of the prohibitive cost of
water filled ditches. Dimensions of moats required for

various species of animals, including large carnivores, are

33

illustrated in the second volume of International Zoo
Yearbook (Anonymous, 1961).

Such water-land barriers, however, are not ideal for
smaller cats simply because it would interpose too great a
distance between the observer's eye and the relatively
small size of the object; also, even a small cat can jump
a considerable distance. Leyhansen (1962) cites an example
Where a serval (E222§_servalL.a long-legged African wild-
cat, from a standing start, twice jumped a ditch 15 feet
wide and landed 4.5 feet above the level on the opposite
bank. For this reason, the improvements in the display of
small cats were unmodified and they are still kept in much
the same way as in the menageries of the Middle Ages.
Leyhausen (22, 222,) has described a most modern ground
plan of a small cat house at the wuppertal Zoo, Germany,
where the minimum area required for a pair of serval or
caracels (E2222_caracal) is 50 sq. yds. of outer enclosure
and 12-15 sq. yds. of indoor cage. A pair of Indian leop-
ard cats (E2222_benga2ensis), however, might do well with
slightly less, but by no means as much less as would be
proportionate to relative size. It is utterly wrong to
think that smaller cats require less space than a tiger or

a lion. If anything, they need more which also holds good

34

both for the indoor cages as well as the outside enclosures.

In designing and constructing animal enclosures, all
practical measures must be aimed at making the animal feel
safe and at home, and at the same time giving it enough
opportunity for movement to keep them busy. In the fittings
of indoor cages, therefore, a variety of tree trunks with
at least some of their branches still intact, pieces of rock
or equivalent, and some relatively broad boards at various
heights on the walls for sitting and resting, should be
liberally used. All these things must be arranged in such
a way as to suggest to the occupant a great variety of
pathways throughout the three dimensional space available.
With a little skill and experience, these can be arranged
giving a maximum sense of protection without unduly ob-
structing the visitor's view.

The semi-natural methods of confining wild animals
include devices such as railings, bars, fencings, and wire
nettings. With advancement in the display techniques,
these devices are being rapidly replaced in many zoological
gardens. In the case of big cats, bars have already been
replaced by wide ditches and moats, or by much lighter,
but sufficiently strong, interlocking wiring. This inter-

locking wire netting has another advantage of rebounding

35

the animal unhurt when, under the impulse of great excite—
ment and escape, they dash against it. Also, it obstructs
the visitor's view less than a fense or a heavy iron bar
cage.

Among the artificial means of confinement, glass and
light seem to be the indespensable items of construction.
Because of its transparency, glass is the ideal material
for most small animals, especially fish, reptiles and am-
phibians. Also, behind glass panes, almost any type of
climate desired can be produced. In addition, glass seems
to be the only solution for undesirable feeding by the
public and subsequent dangers of picking up infection.

The widest use of glass, however, is made for the fishes
which, by the possession of their lateral line, are at a
great advantage over amphibians, reptiles, birds, and mam-
mals. This lateral line appraises them of the presence of
the glass from a distance, thus enabling the fish to avoid
direct collision. Other groups of animals, however, have
no visual means of detecting glass panes. In spite of this
drawback, more and more birds and mammals are now being kept
and displayed behind glass. Birds learn by experience and
discover the exact dimension of their flying space by bump-

ing into it. Once they overcome this difficulty, they turn

If l. l il l l l l l l l Ill l l Ill l I f l! l Ill. 1

36

smartly away close to the glass, but in conditions of
stress and excitement such as a chase during breeding
season, may disregard this space barrier and crash at full
speed.

Despite these limitations, birds may get so accustomed
to glass cages that they completely avoid this invisible
barrier. This fact, biologically speaking, provides a
direct solution of the problem of confined space. With
birds fully accustomed to flying behind glass plates it is
possible to remove the panes altogether, without the birds
flying away. This psychological barrier is further rein-
forced by the action of light where the inside of the cage
is kept bright while the space outside, for the public, is
dark. This type of aviary--with no barrier between the
exhibit and the spectator—-has only recently been devised
and is now successfully used in Frankfurt Zoo, Germany
(Anonymous, 1962), wassenaar Zoo (Gerrits and Diedruch,
1962L San Diego Zoo (Anonymous, 1961a), Zoological Garden,
Philadelphia (Anonymous, 1949) and at Basle Zoo, Zurich
(Hediger, 1950).

Methods of exhibiting and caring for great apes in
captivity have also improved considerably during the past

two decades. These methods are based on the knowledge that

37

these animals, being extremely susceptible to cold and .
influenza viruses, and even to tuberculosis which leads to
dangerous and often fatal illnesses, must, above all, be
protected from infections carried by man. A glass screen
erected in front of both indoor and outdoor cages effec-
tively excludes these elements of danger. In the ape house
of the Antwerp Zoo, this partition consists of "Polyglass."
It is a sheet of glass 2 cm. thick to which a similar sheet
1 cm. thick is attached by gluing at the edges. The 1 cm.
space in between the two sheets is filled with dry air.
This device acts as an insulator against heat and noise and
also prevents condensation. The strength of such a sheet
of glass was tested at the Ghent University laboratory where
it was established that it could withstand the weight of a
300 kilo gorilla charging from some distance above the
ground, five feet away (Bergh, 1960). Presence of glass
panes, however, causes light reflection which makes it dif-
ficult for the visitors to see animals behind their own
reflected images. This could easily be prevented by fitting
these panes at slightly tilted angles.

Another great stride toward eXhibiting nocturnal ani-
mals has been made lately by the use of red light. It is

based on the information that the retina of man contains

38

two types of cells involved in visual perception. The
first, which discriminate color and are excited only by
relatively intense light, are called the cones. While the
rods, the second type of cell, are extremely sensitive to
even minute amounts of light, but do not respond to color
as such. Rhodopsin, the photosensitive pigment of the rods,
however, responds differently to lights of different wave-
lengths. Its maximum sensitivity lies in the range of
green or blue—green, falling sharply toward the red end of
the spectrum where sensitivity is almost nil (Walls, 1942).
Red light, therefore, effectively shuts out the sensitivity
of the rods, allowing the man to see clearly by means of
his cones.

This duplicity of visual cells, however, is not shared
by nocturnal animals, who have, exclusively or nearly so,
only the thin and slender rods in their retina. Southern
(1955), for the first time, utilized this knowledge in
watching the behavior of nocturnal animals in his study to
know the predator-prey relationships among birds and mam-
mals. In the course of his study, he also found that bad-
gers and foxes are also blind to red light and he thought
this might be true with most of the carnivores who hunt

during night.

39

In early 1961, the New York Zoological Park started a
series of experiments designed to reverse the normal ac-
tivity cycle of small nocturnal mammals and render them
visible during the day in red light. The success of these
experiments is evident from the fact that today 39 species
of mammals, representing nine orders and 21 families, have
become active by day under this red light activity reversal
program (Davis, 1962). These animals are on display in the
Small Mammal House of the New York Zoological Garden. The
red light room operates on a 12/12 hour cycle, the "night"
cycle beginning at 1100 hours and the "day" cycle at 2300
hours. The 11 o'clock onset of the "night" period allows
for the keepers to clean the enclosures under white light
of the "day." Under red light, a dirty floor is not clearly
visible and makes the cleaning difficult. The principal
advantage of such red light exhibits is that not only does
the public watch the animals moving around, but keepers
also are better able to watch the physical condition of their
charges during normal working hours.

The only objection to this type of exhibition is that,
in some instances, only a very dim colored light is used
which gives rather a gloomy picture to the visitors. In

order to avoid this trouble the Zoological Garden of London

40

is currently experimenting with some small mammals trying
to reverse their daily cycles under normal lighting con-
ditions (Morris, 1962). The assumption is that the daily
rhythm of small nocturnal mammals is based, not upon the
absolute light strength, but on the relative strength of
light present during the day and during the night. If this
is true, it is possible to reverse their cycles by keeping
them in slightly less light during the day, without chang-
ing the quality of light, and giving more intense and bright
light during night. The results have thus far been encour-
aging, and it seems feasible, at least with certain species,
to allow the public to view such animals in much stronger

and more nearly normal lighting.

Problems of food and feeding
Feeding of wild animals in captivity is not a physio-
logical matter alone, but is intimately tied up with their
social and psychological conditions. The knowledge gained
in this field during the past decade has been tremendous
and the results include the increased life span, reduced
mortality, increased resistance to various diseases, com-
plete disappearance of almost all the deficiency and mal-

nutritional diseases, and healthy, good looking animals in

41

most zoological gardens of today. This knowledge has been
derived directly from the scientific feeding of domestic
animals where it is possible to correct most deficiency
diseases by proper feeding. Despite a large variety of
animals in zoological gardens, this is not impracticable,
for the science of nutrition is based on the fact that all
animals, from single cell amoeba to large mammals, need the
same broad groups of nutrients, i.e., proteins, carbohydrates,
and fats. Vitamins and minerals essentially play the same
important roles in almost any species of animal as they do
in humans and their domestic pets.

wackernagel (1961), in his brochure "Modern Methods of
Feeding Wild Animals in Zoological Gardens," cites several
references where the importance of vitamins and minerals is
shown in a variety of wild animals including birds, mammals,
and reptiles. Scheunert (1933) believed that the "star
gazing disease" of young tigers is caused by Vit. B1 defi-
ciency. Sievers (1957) studied in detail the importance of
Vit. A, C, D, and the B-complex group in the periodical skin
shedding of snakes.

Despite vast experience gained in the field of animal

nutrition, the knowledge has not yet been fully exploited

in the development of practical methods of disease control

42

and maintaining healthy animals in zoological gardens.
According to Hediger (1950), this problem has, so far,
"been too one sided, too physiological and often anthro-
pomorphic." Quite often, people fail to realize that the
mechanics of providing high quality diets to zoo animals
are much simpler than one would ever realize. For example,
the ration for all the 1,595 animals of the Philadelphia
Zoological Garden is prepared by just two men, who also pack
and make allotments according to the exhibition area (per-
sonal communication, Freeman Shelly, director, Philadelphia
Zoo). Also, we must get rid of the old belief that most
wild animals are stenophagous or even monophagous. Leopold
(1933) quotes several examples where tremendous varieties
of food are eaten by most game species. Schaller (1963)
listed a variety of food taken by the mountain gorilla in
the wild. He, however, failed to find a single occasion of
gorillas feeding on animal matter, which is quite contra-
dictory to the belief of those who maintain zoological
gardens. According to Freeman Shelly, meat is one of the
indispensable items in the menus of the great apes. Chim-
panzees, according to Goodall and Van Lawick (1963), have
definitely carnivorous tendencies in the wild and, among

others, they seem to prefer monkeys. Killing and tearing

43

of the flesh is done by both the juveniles and the adults.

Quality and qgantity of food

Considering the fact that no animal is strictly
monophagous, careful attention should be given to the
substitute diet. It must contain all the necessary nu-
trients as well as essential vitamins and minerals in
proper proportions. A balanced diet promotes healthy
growth and builds up a strong natural resistance against
infectious and non-infectious diseases. This is evident
from the fact that enteric diseases, bacillary dysentry,
amoebiasis, balantidiasis, strongyloidiasis and esophago-
stomiasis in apes and monkeys of Philadelphia Zoo are
successfully controlled and cured by just routine feeding
(Ratcliffe, 1963). Also, according to this author, the
lung mites of Asiatic monkeys disappear and the old lesions
heal up within a couple years on the present diet.

For convenience, the animals of a zoological garden
may be grouped as herbivores, carnivores, omnivores, and

piscivores.

Feeding the herbivores
Herbivores, especially ungulates from various ecologic

niches, constitute the major collections of most zoological

44

gardens. These are represented by ruminants such as camel,
llama, deer, giraffe, antelope, sheep and goats. Other
members of the ungulates are the elephants, rhinoceros,
tapir, all members of the horse family and the pygmy hippo-
potamus. The kangaroo, and a few rodents such as chinchilla,
also are herbivorous. Hay of good quality should be given
22_libidum to all of these animals, supplemented by a suit-
able concentrate. Hay, besides providing bulk, is a means
of occupying the animal's attention and energy, and prevents
restlessness. It also assists in preventing obesity. The
concentrate mixture is of great importance, as the hay is
usually not a complete diet, and the supply of fresh greens
or pasture is usually limited or absent in a zoological
garden. Below is the composition of a well balanced con-
centrate mixture designed for the herbivorous animals of the

Basle Zoological Garden (waekernagel, 1961):

Ground oats . . . . . 15 Carob bean meal . . . . 4
Ground barley . . . . 27.2 Ground calcium carbonate 1
Ground grain sorghum. 8 Bone meal . . . . . . . 2
Ground wheat . . . . 10 Salt . . . . . . . . . . l
Soya bean oil meal. . lO Magnesium sulphate . . . 0.2
Groundnut oil meal. . 8 Trace elements mixture . 0.1
Linseed meal . . . . 3 Vitamins mixture . . . . 0.5

 

Lucerne meal . . . . 10 Total . . . . . . .100.0

45

The composition of this mixture in terms of proteins, fats,

and other nutrients is:
Per Cent

Crude protein . . . . . l8
Crude fat . . . . . . . 2 5
Crude fibre . . . . . . 6.5
Calcium . . . . . . . 1 2
Phosphorus . . . . . . O 6
This mixture is made into pellets and is given to all
the animals at the rate of 0.5 to 2 ounces per pound of
body weight, depending upon the size and general condition.

A roedeer thus receives about a pound, while four to five

kilograms is correct for a wisent.

Feeding the carnivores

Fresh meat seems to be the indispensable item of food
for carnivores and most birds of prey. O'Connor (1944),
however, claims that lions and other carnivores may be
maintained on the dry mix she recommended for omnivores.
Muscle meat by itself is not completely balanced nutri-
tionally. Supplementation of this meat, therefore, is
justified not only from a scientific point of view but also
from the observation in the wild where the animals consume
the whole of their prey and not just the meat alone.

All meat fed to animals in the Basle Zoo is supple-

mented with a protein-mineral-vitamin concentrate in the

46

proportion of 7 of this concentrate to every 93 parts of
raw meat used. This supplement consists of 9 parts of dry
yeast, 9 parts of skimmed milk powder, and 3.5 parts of
mineral-vitamin mixture (Wackernagel, 22, 222,). It is
mixed either with the minced meat or sprinkled on the pieces
of meat as they are fed to the large beasts of prey. Be-
cause of its palatability, it is liked very much by all the
animals; also it adheres well to the chunks of meat. This
feed is given to mammals and owls and to several other
birds of prey.

The meat supplement for carnivores in the Philadelphia
Zoological Garden consists of oystershell flour 50 parts,
powdered skimmed milk 45 parts, and iodized salt 5 parts.
This is mixed with raw meat in the quantity of 12 to 13
parts of the supplement to every 86 or 87 parts of the meat
and 2 parts of Vit. A-D feeding oil. This is given at the
rate of 0.5 to 2 ounces per pound of body weight (Ratcliffe,
1963).

For adult members of the Felidae, raw meat (preferably
horse meat) with 1—4 ounces of the above supplement is
given at the rate of 20 to 100 gm/kg of body weight.
Leopards, Cheetahs, and jaguars are fed a whole chicken (2

to 3 lbs.), with bones, once or twice each week. Smaller

47

cats are given pigeons or ducklings once or twice a week.
This change of diet is necessary in maintaining an animal's
interest in food and also to improve its appetite. Raw
liver acts as a laxative and stomachic and is relished by
most animals. It is always given when animals are consti-

pated and subsequently lose their appetite.

Feeding the 22scivores

Fish-eating animals like sea lions, penguins, pelicans,
and cormorants present a different situation. Fish, which
are commercially obtained, may not be adequate for these
animals, but according to Ratcliffe (22, 222,), there is no
practical solution by which fish may be improved. Zoos,
that are able to obtain oily fish, like herring and mackeral,
however, report better results with fish-eating animals.

Penguins present a constant problem to most of the zoo—
logical gardens. They suffer from a special type of intes-
tinal catarrah almost always accompanied by mycosis.
Sladen's (1954) observation of penguins in the wild and in
captivity indicates that these animals are remarkably free
from disease in nature and it is only when they are brought
into captivity that they become susceptible.

wackernagel (22, 222,) believes that food-fish for the

animals may, during transportation, lose some micronutrients

48

due to decomposition and produce toxic substances causing

intestinal catarrah and subsequent mycosis. He, therefore,
suggests to fortify the fish diet and treat the fish with
a bacteriostat. For this the fish on arrival is submerged

into a solution of acromize (chlortetracycline) and stored

on ice. His mineral-vitamin mixture consists of:
Standard mineral mixture . . . . . . . . 5 kg
Vitamin preparation . . . . . . . . . . 860 gm
The above vitamin preparation contains:
Vitamin A . . . . . . . . . . 3,000,000 IU/kg
Vitamin D2 . . . . . . . . . . . 600,000 IU/kg
Vitamin E . . . . . . . . . . . 60,000 IU/kg
Vitamin B1 . . . . . . . . . . . 700 mg/kg
Vitamin B2 . . . . . . . . . . . 2,300 mg/kg
Nicotinic acid . . . . . . . . . 9,000 mg/kg
Calcium D-panthothenate . . . . 3,500 mg/kg
Vitamin B6 . . . . . . . . . . . 1,200 mg/kg
Folic acid . . . . . . . . . . . 300 mg/kg
Vitamin B12 . . . . . . . . . . 35 mg/kg
About 5 grams of the above mineral-vitamin mixture is put
into a gelatin capsule and fed to penguins and sea lions at
the rate of one to two capsules per animal. These capsules
are put into the fish before being offered and are thus

readily taken.

Feeding the omnivores
The diet developed at the Philadelphia Zoological

Garden for omnivorous animals seems to be simple and quite

49

adequate for a variety of animals. The basic diet consists
of the following ingredients (Ratcliffe, 22, cit.):

Ground yellow corn . . . . . 15

Ground whole wheat . . . . . 15

Ground whole barley . . . . . 10

Ground rolled oats . . . . . 10

Peanut meal . . . . . . . . . 10

Soybean . . . . . . . . . . . 10

Alfalfa meal . . . . . . . . 5

Brewer's yeast . . . . . . . 10

Dried skim milk . . . . . . . 10

Oystershell flour . . . . . . 2

Iodized salt . . . . . . . . l

A—D feeding oil . . . . . . . 2

100

This mixture is prepared in bulk but never stored for more
than two weeks. Nine parts of this are combined with one
part of ground, boiled meat and sufficient meat broth or
water to make a stiff mash. This is thoroughly mixed,
pressed into shallow pans, refrigerated for 24 to 48 hours,
and cut into pieces for feeding. Ordinarily, it is supple-
mented by oranges and carrots for apes, and carrots, kale
or escarole for monkeys, baboons, and other animals. This
ration supplies approximately four calories per gram and is
given at the rate of 1 to 10 g/kg of body weight, depending
upon the age, care and condition of the animal. This ration,
with or without slight modification has been found to be

completely adequate for the following mammals and birds at

the Philadelphia Zoological Garden:

Family

Pongidae

Cercopithecidae

Lemuridae

Cebidae

Hylobatidae
Callithricidae
Castoridae
Muridae
Cricetidae
Chinchillidae
Hystricidae
Erethizontidae
Ursidae
Procaviidae
Suidae
Tayassuidae

Bradypodidae

50

Mammals

Gorilla, chimpanzee, orangutan

Baboons, Asiatic monkeys including
langurs, African monkeys including
colobus

Lemur

Cebus, spider, squirrel, and wooly
monkeys, saki, uakari

Gibbon

Marmosetl

Beaver

Rats and mice
Hamster

Chindhilla

Old world porcupines
New world porcupines
Bear

Hyrax

Swine

Peccary

Two-toed sloth

 

1For debilitated apes and monkeys, equal parts of this
ration are mixed well with equal parts of carnivore ration
(page 46) and finally ground in a meat grinder.

2

Half of this ration is mixed with equal parts of

carnivore ration (page 46), fish, suet, and carrots. For
the polar and kodiak bears, 4 to 8 ounces of peanut oil is
added to the carnivore diet.

51

The omnivorous diet discussed on page 49 has been

found completely adequate for the following birds:

Family

Anhimidae
Anatidae

Gruidae
Rallidae
Columbidae
Tetraonidae
Numididae
Meleagrididae
Struthionidae
Dromiceiidae
Rheidae
Casuariidae
Phoenicopteridae

Psittacidae

Birds

Screamers

Ducks, swans, and geese except
those that eat only fish

Cranes and their allies
Coots, gallinules
Doves, pigeons
Grouse

Guinea fowl
Turkeys

Ostriches

Emus

Rheas
Cassowariesl
Flamingos

Parrots, parakeets, lories, lori-
keets, macaws, keas, conures,
cockatoos and cockatiels2

 

1Equal parts of omnivore and carnivore diet.

Larger psittacines are fed on a mixture of 2/3
omnivore ration and l/3 carnivore diet. For small lories,
cockateels, and parakeets, half of the omnivore ration is
mixed with 20 parts of ground raw carrots, 20 parts of
ground cooked meat, 8 parts of ground hard-cooked eggs
and shells, and 2 parts of A-D feeding oil. The final

ration, thus prepared,

is mixed with some seed, otherwise

birds do not seem to recognize this ration as food which
may reflect their innate behavior pattern.

52

For flamingos, 90 parts of the omnivore ration and 10
parts of ground boiled meat are mixed thoroughly and mois-
tened with fresh pulverized carrots and carrot juice (1
gallon to 20 pounds of dry weight), refrigerated and used
within 6 hours; 0.5 to 0.75 pounds of the ration divided
into morning and evening meals is given to each bird per
day.

Freshly ground carrot and carrot juice are essential
for retaining plumage colors of scarlet ibis, flamingos,
and other birds having carotenoids (red, yellow, and orange
pigment) in their feathers. In the Basle Zoo, flamingos
are provided with l per cent of fat-soluble extract of

lucern to preserve their colors.

Social and psych22ogical problems in feeding

Feeding wild animals in captivity, as said earlier, is
not simply a physiological matter, but is closely bound up
with the animal's social and psychological state. Flight
reaction is the most significant characteristic of the wild
animal's life in freedom, and it must be given prime con-
cern in captivity, especially with newly captured ones.
With untamed animals, flight comes before feeding.

Feeding freely and satisfactorily in the presence of

man is one of the most important proofs that an animal has

53

settled down. Many animals, from fish to mammal, have been
known to go on hunger strikes in reaction to captivity.
Brown (1932) cited such an example in an aard-wolf which
did not eat or drink for several days when first brought to
Philadelphia Zoological Garden. Hediger (1950) recommends
that in such situations a restful atmosphere with lack of
disturbance and darkness is quite helpful. Force should
always be avoided as it only aggravates the animal's
excitement.

It is a common experience that the amount of food eaten
by an individual can be greatly increased if fed in the
company of other members of the same species, or of other
animals accepted in their social organization (animal's
assimilation tendency). The influence of this companion-
ship factor can be traced through all the vertebrate king-
dom, from fish to mammal.

The effect of social factor on food intake has been the
subject of intensive researches, especially on hens. Katz
(1937) states that under the influence of the social factor
the food intake in chidkens can be increased over 60 per
cent. Conversely, isolation of individual chimpanzees from
a social group caused fasting for several days. In the

best interests of the animal, therefore, it is essential

54

not to keep them isolated. Lang (1960) said that all great
apes are social animals, and it is important not to keep
them isolated. Especially when young, they should be
brought up in the company of their own kind, or of a closely
related species. Slow orangs and gorillas, for example, are
favorably influenced by the lively chimpanzees.

Another important aspect of the biology of zoological
gardens is the feeding pattern in different animals.

Hediger (1950) distinguished between two extreme types of
feeding in zoo animals. These are the continuous and the
occasional feeders which are found, again, throughout the
animal kingdom, from fish to mammal. The continuous feeders
are represented by plankton-eating fish, toads, tortoises,
passerine birds, rodents, ungulates, elephant, brown bear
and monkey. Examples of occasional feeders are predatory
fish, snakes, birds of prey, and great cats.

Intake of food, like any other biological activity of
the animal, is part of its space-time pattern. The free-
living giant panda (2222ropus melan22eucus), for example,
spends from 10 to 12 hours to take sufficient food. It
wanders along its habitual tracks in comparatively small
territory of about one square mile, eating on bamboos,

which are relatively poor in nutrition. Apparently, the

55

chief feeding period is at night and thus the food intake,
in this animal, is spread over time and space. Another
extreme example perhaps is found among snakes which await at
a particular spot until a victim appears. In a few moments
this is captured, swallowed, and the animal retires until
the victim is completely digested.

These occasional feeders react quite differently to the
food stimuli than do the continuous feeders (bear, sea-lion,
elephant). This power of food attraction, therefore, may
be used for drawing the animal's attention and increasing
the intimacy of the animal-man relationship. Continuous
feeders, while training, must be rewarded for accomplishing
every single performance with food such as sugar for bears,
and fish for sea-lion. Training sea—lions without fish, and
bears without sugar, says Hediger (22, 222,L is unthinkable.
In occasional feeders like lions and tigers, however, food

stimuli play no part at all.

Exercise and training

Considering the fact that wild animals are, in nature,
Constantly engaged with the impulses to avoid enemies and
to seek food, and that both these highly significant

elaments suddenly disappear in captivity, it is obvious

56

that the change must have some far reaching consequences.
Since the captive animal's most important occupations are
taken away from him, enormous amounts of energy are thus

released, and these must somehow be utilized.

Apparently, the most immediate problem in the biology
of zoological gardens arises from the want of occupation.
Lack of engagment may have some harmful results, manifest-
ing itself in various ways. Some of these secondary effects
of captivity appear in the form of excessive anti-social
behavior, hypertrOphy of sexual activity, and stereotyped
movements. The animal, in order to break through the
boredom of life, resorts to several means of self amusement
and to subsequent vices. These, in turn, may lead to such
symptoms of captivity as reduced body size, muscular
dystrophy, lighter skeleton etc. The captives, therefore,
must be given a new interest in life, an adequate substi-
tute for the chief occupation of freedom. A biologically
suitable training, which also provides a sort of occupa-
tional therapy, seems to be the best solution.

Zuckerman (1932) believes that regular provision of
food in zoological gardens releases energies in the animal
which, in the absense of proper occupational therapy, are

used up probably in more intense sexual behavior. This is

57

much more so in those animals which breed continuously, or
nearly so, the whole year around (e.g. giraffe and anthro-
poid apes). Since sexual behavior is closely connected with
social behavior, this hypersexuality has far reaching effects
in the biology of zoological gardens. Hypersexuality in
captives makes possible an increase of socially conditioned
fighting where the social rivals cannot get as far away
from one another as in nature. Thus, after a fight, both
the victor and the defeated partner still cannot lose sight
of each other, and this state of affairs often leads to the
conflict never being settled as long as two rivals live
together. In freedom, the animals separate and can thus
avoid a final solution of the conflict. But the only real
solution in captivity seems to be the death of the rival.
Zuckerman (22, 222,) gives several examples of serious
losses from socially conditioned fights of this kind in a
colony of captive hamadryad baboons (Papio hamaggyas). The
chief conclusion to be drawn from this fact is that it is
often more suitable, and more correct biologically, to keep
those species which fight under social stimuli in smaller,
harmonious groups, and keep them busy by providing suitable
training exercises. Naturally, the more highly organized

is the captive animal, the more its lack of activity will

 

58

be noticed. No wonder, then, that training exercises were
first tried out on apes.

Training, however often it is used as a scientific
auxiliary, was not a laboratory invention. It represents
an age-old way of managing animals. It was used by primi-
tive men, as it still is used intuitively by the trainers
in the modern circus. Training as a scientific aid, as far
as the fundamentals are concerned, is not even a century
old, but these have certainly been borrowed from the circus
or its forerunners. Hediger (1950; 1955) distinguished all
the intermediate stages between freedom and domestication
through which an animal has to pass in the course of being
tamed or trained. Training, according to Hediger (1938),
represents a transitional stage through which the animal
passes to reach the fullest kind of relationship with man;
the state of being completely trained.

In scientific training, the elimination of the animal-
man relationship is at its maximum while in a circus this
relationship is at its extreme intensification. This is
quite natural considering the amount of money circuses spend
on staff and the type of performance required from their
animals. In circuses nearly every animal is almost con-

tinually in close contact with his trainer, the riding

59

master, the groom, and the watchman. In the zoos, however,
each keeper is assigned to a large area, covering several
animals, and he has little or no time to spend on separate
individuals. Zoo training, therefore, should be limited to
simple exercise training in the best interest of the animal,
as a part of occupational therapy; performance of high
standards and sensational Showmanship should be left to the
circus.

The need of occupation evidently is found not only among
apes but in many other species, for example sea-lions, many
predatory animals, ungulates, birds, and even certain rep-
tiles and fishes. Hornaday (1936) listed the following
species, in order of their importance, whose zoological park
training is a matter of necessity: elephants, bears, apes,
hippopotami, rhinoceroses, giraffes, bison, musk-ox, wild
sheep, goats and deer, African antelopes, wild swine and
wild horses, asses and zebras. Of large birds the most
conspicuous candidates for training are the ostriches,
emus, cassowaries, cranes, pelicans, swans, egrets and
herons, geese, ducks, pheasants, macaws and cockatoos,
curassows, eagles and vultures. Among the reptiles, the
best trained are the giant tortoises, the pythons, boas,

alligators, crocodiles, iguanas and gopher snakes.

60

In most zoological gardens, sea-lions are seen taking
hold of a piece of wood floating in their pond, tossing it
up and catching it again. Many predators, too, appear to
enjoy interruption of their monotonous life; and for this
they are often provided with big wooden balls for play.
This also causes regular wear and tear of their claw nails
which otherwise overgrow and cause sore pads.

Many zoo animals that are kept in spacious enclosures
can sometimes find their own specific games at will.
"Mohan," the chimpanzee at the Calcutta Zoological Garden,
for example, had a small table and a chair. Every day
during lunch hour he would set his dining table and chair
properly on the lawns of his island, sit straight on the
chair, and would start his lunch from the bowl and the plate
brought him by the keeper. After the meal, he would take
the table and chair back to their position and would in-
dulge in all sorts of acrobatics, including swing and
horizontal bar exercises.

Through play and training, contact between man and
animal is so intensified that often man is eventually
accepted as a member of the species, and incorporated into
the social structure of the group. This intimacy often has

several practical advantages. The above mentioned

61

chimpanzee of the Calcutta Zoo, for example, once suffered
from a bad type of intestinal tuberculosis. The keeper used
to bring the prescribed medicine to him in paper folds. The
chimp would voluntarily accept the medicine, open it up, put
it into his mouth, and chew up the whole thing without los-
ing even a single grain. The result was a quick and complete
cure without much trouble. This is quite unnatural as it has
often been observed that no ape in captivity would eat even
the best food offered to him once he has the slightest hint
of some medicine mixed in it.

In addition, the material value of an animal, or of a
group of animals, can be greatly increased through training
because groups of trained animals have more show value than
the untrained ones.

Morris (1961) has recently described an automatic feeder
"Mark II Seal Rotor'I for the gray seal of the London Zoo.
These seals are housed in a large circular pool with rails
installed around its rim, on which travels an electrically
driven box with a capacity to travel up to 30 miles an hour.
A fish—release button opens a series of flaps on the under-
side of the box as it moves around the pool, which could be
pressed at any point in the run. This makes it impossible

for the seals to learn exactly where the fish will appear,

62

and forces them to follow the box competitively, in order
to be the nearest one when a fish falls into the water.

In the improved variety of the "Rotor," the fish, in-
stead of falling in the water, simply hang in a group from
traveling arm. The seals, thus, have not only to chase
after the box, but also have to leap repeatedly out of water
to snatch the fish. This rotor, in addition to providing
the seals with plenty of exercise, has become a major
"feeding time" attraction for the public.

The London Zoo is currently experimenting to improve the
design of this apparatus for a variety of species in which
the animals will be required to perform various activities

to obtain food.

Disease contro2 and animal restraint

If wild animal reserves are to be built up, both to
maintain the disappearing species and to provide a protein
bank for the world's ever-growing population (Halloran,
1955), it is essential that scientists should learn more
about the general welfare and disease problems of these
animals. The popular conception that wilderness areas
produced a healthy lot of animals is quite mistaken. As a

matter of fact, there is not a single group of animals that

63

is spared. Captivity, however, through numerous physical
and mental derangements, often aggravates the situation.
Unfamiliar, unnatural and unvaried food, change of climate
and environment, physical and mental degeneration from
disuse of muscle and brain, fear, ennui, nostalgia, lack
of exhilaration of chasing and being chased, often unsatis-
fied sexual desire--all comprise a complex social and bio-
logical environment for the animal and react harmfully on
the captive. To study the actions and interactions of all
these elements in the vast array of the animal kingdom, a
zoological garden provides the best laboratory.

During recent years, the interest taken by the veteri-
nary profession in the diseases of non—domestic animals has
been tremendous. Halloran (22, 222,) published a connota-
tive bibliography of references to diseases in wild mammals
and birds, both feral and captive, obtained from over 900
different publications throughout the world, covering a
period of 120 years (1830-1950).

Graham-Jones (1962), in his concluding remarks on the
4th International Symposium on diseases in zoo animals, said
that the reasons for these rapidly growing interests are:
economic, ethical, a healthy competition among zoos to

exhibit contented and disease free stock, and the study of

64

various diseases mutually communicable between man and ani-
mals. This last impetus, thus, has led to establishment of
various institutions of comparative medicine and comparative
pathology throughout the world.

Since the time of Darwin, who systematized the world's
knowledge of comparative biology, pathologists have been
trying to explain the abnormalities in the light of Darwin's
principles. Bland-Sutton's work, "Evolution and Disease,"
as early as 1890, was probably the first step in this
direction.

On the occasion of the celebration of the 50th anni-
versary of Darwin's great work, "Origin of Species," ilya
Metchinikoff, in his address at Cambridge, explained how the
idea of evolution stimulated the discovery of phagocytosis
and the development of the comparative pathology of inflam-
mation. The study of evolution has proved that all living
beings on earth are related to one another through origin
from common ancestors. It presents an obvious basis for the
suggestion, then, that certain pathological processes may
appear in their most primitive forms in lower organisms,
where their expression is particularly favorable for analysis.

Huxley (1958), in his book, "Biological Aspects of Can-

cer," strongly defended the idea that cancer is not merely

65

a medical problem; it is a biological phenomenon, and its
elucidation is closely connected with advances in a number
of key branches of present—day biology. Despite these
realizations, the application of evolutionary principles to

the practical problems of pathology is still in its infancy.

322e of veteginary services in a zoolggica2jgarden

The importance of veterinary services in the biology
of zoological gardens can be justified for two reasons:

a) Maintenance of good, healthy looking animals free
from disease or disease carrying agents. This includes the
therapeutic as well as the preventive medicine.

b) Applying scientific methods of animal restraint in
the day to day zoo—husbandry work. It includes both the
mechanical and the chemical restraints such as sedative,

anaesthetic, ataractic, and tranquilizing drugs.

Problems of disease and disease pggyention

In the absence of any systematic guide to the diseases
and treatment of wildlife, disease prevention is probably
the prime concern of a veterinary program in a zoological
garden. Diagnosis of disease in a wild animal, except of
course those which are self-evident, is more often than not

speculation and conjecture. It is not uncommon for animals

 

66

to come to autopsy presenting a galaxy of abnormalities,
yet the antemortem observation may fail to reveal any un-
usual appearance. On the other hand, some specimens are
opened which fail to reveal lesions even on close and
careful examination. Careful observation, however, may
sometimes lead to correct diagnoses.

Physical examination is limited to tractable beasts and
to those which can be caught and handled without danger to
personnel or unusual fright and damage to themselves.
Anaesthetics and sedatives are not always practicable,
especially when the animal is of bad temperament and/or
seriously sick. Animals do not like to be molested; on
the other hand they seem to enjoy attention. Whenever
possible, handling should be avoided or done under tran-
quilizers. A discussion about the usage of tranquilizing
drugs will appear in the forthcoming pages.

Effective cure of any illness depends on early detection
and prompt care. It is important, therefore, that signs of
illness or troubles be regularly watched by the keeper in
charge. Some of the common symptoms to be looked for are
lack of appetite, weight loss, lack of grooming, inactivity,
dullness of eyes, unusual falling of hairs, looseness of

bowels or constipation, convulsions, coughing, excessive

 

llllllllllll

 

67

thirst, discharge from eyes or nose, and rapid or labored
breathing. Appetite, as usually considered, however, is
not always a reliable guide to the state of health. Many
healthy animals refuse food through physical excitement
and sick ones often take it until they collapse and die.

Another reason that poses far more difficult problem
for the veterinary surgeon is the lack of acquaintance with
the anatomical and physiological organization of wild ani-
mals. Knowledge of these disciplines is indispensable for
any therapeutic measures since some animals show marked
differences from closely related species. Often the knowl-
edge of domestic species is applied to animals of similar
physiology. Analogies, for example, can be made among
canines, felids, and mustelids, yet perplexing differences
are common.

Because of these and many other difficulties, treat-
ment of wild animals 222.§2_is not at all encouraging. It
is essential therefore to collect all the information
available from the field and to make it readily available.
A close cooperation on an international scale is desirable.
A passive role, in.the form of control and prevention of
disease, therefore, should be the major objective of the

veterinary services in a zoological garden.

 

llll’lt‘l

68

The importance of a scientific, balanced diet for the
control and prevention of disease can scarcely be over-
emphasized. Carefully designed diets certainly should be
an essential feature of the environment of every zoo; first
because the more obvious malnutritional diseases are thereby
eliminated, and second, because adequately nourished animals
develop and maintain high levels of resistance to many
microorganisms that otherwise induce disease.

After the introduction of complete, balanced rations in
the Philadelphia Zoo, one of the effects observed was a
marked decline in the incidence of tuberculosis among birds
(Ratcliffe, 1958). This has been a helpful factor in the
development of resistance to tuberculosis, especially in
the waterfowl collection (Ducks, geese, and swans). Simi-
larly, enterocolitis of apes, monkeys, and baboons, which
is a common affliction of newly imported animals, has been‘
cured by appropriate diet. The carrier status usually per-
sists for a time, but eventually the agents of disease are
eliminated within a year or so without resort to specific
therapy. The diet, likewise, has offered effective resist-
ance among ruminants and non-ruminant herbivores against
nematode parasites, including stomach worms (Haemonchus)

which have been a common cause of mortality among these

animals.

69

In addition, the introduction of a scientific, well
planned feeding system makes obvious other limiting factors
concerned with the successful keeping of animals. Thus, if,
despite a complete ration, the condition of a species is
unsatisfactory, then more attention must be paid particu-
larly to their social factors, and also to the sanitary
conditions of the pen and all of the animal's surroundings.
Ratcliffe (1962), for example, listed 18 per cent of all
deaths among mammals (representing primates, rodents, felids,
bovids, and marsupials) as due to arteriosclerosis. This
was evenly distributed among males and females, ranging in
age from 2 to 245 months. Analyzing the situation in the
light of other pertinent information, he came to the con-
clusion that environmental factors, especially the social
environment (number, sex, age ratio), are a major factor in
the development of this disease. In order to study the
effects of social factors on the development of athero—

_ sclerosis a separate experiment was designed which is dis-
cussed in Part II of this thesis.

Control of infectious diseases in a zoological garden
is highly important since there is reciprocal danger of
Ipicking up infection from man to animal and vice versa.

Fox (1923) , for example, mentioned that tuberculosis has

70

not with certainty been diagnosed among animals living in
a wild state, unless they have been in contact with human
beings. Among zoo inmates, primates suffer most and the
public is the source of infection. With very few exceptions,
an airborn infection acquired through exposure to open
cases of tuberculosis is the rule. However, with the im-
proved management and display techniques, the disease has
now practically been controlled. The glass partition
erected in front of both the indoor and the outdoor cages
effectively does away with the possibilities of infection
passing from man to animal or from animal to man. Among
other members of the primates, potentially dangerous to
humans, are the gibbons and chimpanzees. Gibbons are fre-
quently carriers of the shigella dysentry organism, and
chimpanzees, of Entamoeba histolytica, 222antidium, and
other parasites which live in the alimentary canal.
Recently, chimpanzees have also been shown to be healthy
carriers of the virus of infectious hepatitis, a very
serious disease of adult humans (Fiennes, 1962).

Another indispensable measure in the control and eradi-
cation program of tuberculosis in apes and monkeys is the
tuberculin test combined with prOper quarantine. The

method, by keeping a record of rectal temperature both

'1' ‘ III' I l I, ll III
IIIle l l ll l l
I I Ill I ll Ill I I'll. I III ll —

71

before and after the injection, has been fully described by
Fox (1923). The procedure, although expensive in terms of
time and personnel, is quite simple. However, temperature
is not always a reliable guide as it may shoot up due to
excitement.

The palpebral test, originally described by Kennard 22_
22, (1939), is now commonly used in both large and small
animal practice. The procedure for this test in apes and
monkeys, after Ratcliffe (22, cit.) is as follows:

Restrain the animal by hand or in a net and inject
into the upper eyelid 10—15 mg. Old Tuberculin/kg. in

1 ml. or less of fluid. Redness and swelling sufficient

to partially or completely close the eye usually develop

within 24 hours and persist for 72 hours if the animal
has tuberculosis. Non-tuberculous monkeys are not
affected.

If some positive animals are found in a group of quar-
antined animals, the healthy ones are still kept isolated
and retested after two months. These are passed for exhi-
bition only when they prove negative on the second test.
This test, coupled with a periodic X-ray examination of the
keepers and attendants and an effective glass barrier between

the visitors and animals, have been proved to be most effec-

tive in the control and eradication of tuberculosis.

 

72

Pro222ms ogianima2_£estraint

Those responsible for day-to-day management of captive
animals are fully aware of the problems associated with
getting near an animal, without either provoking the ani-
mal's flight reaction and causing alarm and injury, or im-
posing upon themselves a greater danger than ordinarily is
sensible. Knowledge of the techniques of controlled hand-
ling of wild animals is essential for their protection and
welfare. Some of the aspects of zoo management which have
been assisted by proper restraint of animals may be listed
as:

a) Animal management: The problems of breeding and
rearing are complicated sometimes by viciousness that can
be controlled by modern drugs. Introduction of new mates,
and acceptance of newborn by the mother, are frequently made
easier (Gandal, 1962).

b) The shipping, crating, and transit of wild animals,
previously a big task, is now greatly assisted by the use
and development of modern tranquillizing and ataractic drugs.

c) The treatment of many diseases is assisted by the
use of modern sedative drugs either indirectly to make the
patient more amenable, or directly, if the illness is asso-

ciated with a neurosis.

"' a“

:fi . .1

 

 

73

Mechanical devices like squeeze cages, crates, nets,
and moving boxes, although indispensable items in the man-
agement of a zoological garden, are very limited in their
range. These are resisted by force, and the animal may use
its teeth, hands, and feet for defense. Furthermore, apes,
monkeys and many more animals seem to possess a remarkable
memory, and an imprint upon an animal of an unpleasant
situation may make it more apprehensive of any repeat.
Physical restraint, especially in primates, therefore is
always undesirable.

Recent advancements in tranquillizing and ataractic
drugs have opened up a fascinating field of wild animal
restraint, both in the feral and in captivity. Successful
immobilization of a variety of animals has been achieved in
game reserves and in zoological gardens.

A standard solution of succinylcholine chloride (20
mg/cc) is extensively used to cause temporary paralysis
lasting anywhere from 10 to 40 minutes. The drug is a
neuromuscular blocking agent and blocks the transmission
of the nerve impulse at the neuromuscular junction, the
place where the nerve and muscle join. The action of the
drug is accomplished chiefly by causing a persistent de-

polarization of the muscle and plates. Since the effect of

74

the drug is only on voluntary muscle, and since the muscles
of respiration fall into this category, respiration is
seriously affected by doses sufficient to cause paralysis.

Heuschele (1961) used this drug on 44 different animals
(lion, tiger, bear, sea-lion, deer, elk, and hyena) at the
San Diego Zoological Garden using a projectile syringe in
which seven animals were lost and five were not affected at
all. He found a considerable species variation with respect
to drug dose per pound body weight required to produce com-
plete immobilization and with respect to the margin of
safety of the drug. This means that careful experimentation
with each species must be carried out to ascertain the safe,
effective dosage limits for it. In handling highly nervous
and fractious animals it is advisable to use tranquillizing
drugs 1 to 2 hours prior to immobilization.

Another drug extensively used in the zoological gardens
is chlorpromazine hydrochloride ("Largactil," May & Baker).
This has been found more suitable than any other ataractic
drug chiefly because it also has a soporific value which is
of advantage in conditioning newly caught animals. Dosage
range from 1 mg./1b. body weight for antelope to 0.125 for
fully-grown giraffe. Harthoorn (1961) used this drug on 30

black rhinoceroses and on a number of giraffes and zebras

75

for transportation without fatality. He further indicates
that dosage rates for animals in zoological gardens seem to
differ markedly from those advocated for free roaming ani-
mals. This may be due to the interactions of various fac-
tors working on animals in captivity which lower their
resistance to such drugs. Dosage for most species is still

unconfirmed and considerable related research is needed.

Interaction of public and zoo tenants

From the preceding discussions it is apparent that the
public is the reason for the very existence of most zoolog-
ical gardens. Experience, however, has shown that they also
provide the greatest source of danger to zoo tenants. The
duty of a zoo biologist, thus, lies not only in taking
appropriate measures to protect the public from the animals,
but also the other way around.

Visitors may become a source of danger to animals for
three reasons. First, by carrying infective agents of dis-
eases that are readily communicable from man to animals.
This aspect has already been discussed in the section on
disease control. A protection such as the glass partition

provides the most effective and practical solution.

The second serious danger lies in offering unsuitable

76

food to animals. Appelman (1961) indicated various prob-
lems involved in imposing a complete ban on such feeding by
the public. He made a careful record of the various items
and quantities of food given by the public to certain
specific animals. Results of his observation indicate a
clear behavioral difference among different species. The
elephant, for example, virtually accepted and swallowed
everything given to him without discrimination. The gibbon
accepted everything, but ate certain foods and rejected the
rest. The zebra, on the other hand, accepted only those
items that seemed edible to him, but refused foods with a
strong or strange smell. Below is the list of items offered

to and eaten by an Indian elephant:

 

 

Items Offered as Food Quantity Offered
Peanuts . . . . . . . . . 1,706
Slices of bread . . . . . 16 with ham

11 with cheese
28 with chocolate
14 with sugar

Sweets . . . . . . . . . . 1,330

Ice cream . . . . . . . . 2 whole
5 pieces

Sugared biscuits . . . . . 811

Apples . . . . . . . . . 17

Pieces of paper . . . . . 198

Paper bags . . . . . . . . 3

Lady's white leather glove 1

Boot lace . . . . . . . . 1

77

From the list of items given, one can realize how
disastrous offered food may be to the health of the animals,
especially to those which accept and eat most everything
offered to them.

Dobberstein (1936) reported instances of serious in-
digestion with fatal results, among animals of the Berlin
Zoo after days when there had been large crowds. Quite
often people fed tainted and spoilt food, perhaps not
realizing that these could cause just as serious an illness
in animals as in man. Hediger (1950) cited an example
where a polar bear contracted severe haemorrhagic gastro-
enteritis after it was fed putrid sea fish by a woman
visitor who had brought it expressly for this purpose.

Some zoos allow the public to feed certain animals.
This expedient, however, does not seem to be a practical
solution to the problem. People continue to bring all
sorts of foods and are careless as to which animals they
are permitted to feed. A complete ban on feeding zoo
animals by public, as decided by the International Union
.of Zoo Directors at Copenhagen (Appelman, 22, 222,), seems
to be the only real solution.

The third and the most serious danger lies in the

possibility of direct harm or indirect damage to the animals

78

by worrying, teasing, and frightening or exciting them.
Innumerable cases are on record where people, from malice
or morbid disposition, have deliberately maimed or killed
valuable animals. Dobberstein (22, 222,) cited several
examples of malicious poisoning among young bears in Berlin
and other zoological gardens of East Germany. Hediger (22,
222,) reported the tragic death of the two-ton, giant sea-
lion of the Hamburg Zoo in 1930. In the stomach of this
victim was found a broken beer bottle which caused per-
forations and fatal peritonitis. On another occasion, the
glamorous bill of a whale-billed stork (Balaeniceps rex)
was smashed with a hammer. In still another case, an ele-
phant at the Geneva Zoological Garden (now closed) was
offered an apple in which several pins were concealed.
Records show that no zoo in any part of the world is safe
from such regrettable incidents.

The most nearly practical solution of the above problem,
as it appears to me, lies with the zoo personnel. The
people that visit zoological gardens must be taught a new
attitude towards animals. A zoo fits into a niche in the
community in a number of ways. Zoological gardens of today
are not just a place of amusement, but a biological insti-

tution. People must be taught a truer, more positively

79

bio10gical attitude towards animals. The result depends
largely on how it is presented to the public and how it
fits into the existing educational program of the community.
The modern zoological gardens, besides showing animals,
have to show them in relation to time and space; not the

animal as an object, but the animal as a subject.

PART II

EXPERIMENTAL ATHEROSCLEROSIS IN GROUP AND
SOCIALLY ISOLATED JAPANESE QUAIL
(COTURNIX COTURNIX JAPONICA)

INTRODUCTION

Studies of the past decade have shown that social
interactions in populations of mammals and birds have pro-
found effects on behavioral phenomena, physiological proc-
esses, responses to drugs, and susceptibility to a variety
of diseases. With respect to behavior, interpreted as a
measure of emotionality, it has been consistently reported
that group-living animals are less emotional, timid, or

fearful than animals caged individually (Stern t 22,,

1960; Ader _2__2,, 1963; Ader and Friedman, 1964). The
data, however, are not so consistent with respect to re-
sistance to disease. Relative to animals caged individu-
ally, the group-housed animal is more susceptible than the

individually—housed animal to alloxan diabetes (Ader t 22,,

1963), whole—body X-irradiation (Ader and Hahn, 1963),
trichinosis (Davis and Read, 1958), and immobilization-
produced gastric ulcers (Stern 22_22,, 22, 222,). These
observations, however, are contradicted by others who have
shown that animals caged in groups are more resistant to

spontaneously developed mammary tumors (Andervont, 1944;

t al., 1955), and

Muhlbock, 1951), convulsions (King

conflict-induced gastrict erosions (Conger 22_22,, 1958).

82

Over the past three decades, death records of the
animals at the Philadelphia Zoological Garden indicate an
increase in the occurrence of arteriosclerosis in mammals
and birds. This increase is characterized by two distinct
bounds. The first increase was observed following 1935,
when the scientific, balanced diet was first introduced.
Since then, the diet was held constant. Nonetheless, a
second increase occurred with a change in distribution of
the lesions (Ratcliffe and Cronin, 1958; Ratcliffe _2_22,,
1960). This second increase was attributed to stress and
anxiety as a result of changes in each animal's environ-
ment, namely, an increase in cage population, noise, and
frequency of contact with man (Ratcliffe and Snyder, 1962).

Katz _2_22, (1958), emphasizing the nutritional-
metabolic cholesterol-lipid-lipoprotein theory of athero-
sclerosis, have said that diet does play a decisive, but
not an exclusive, role in the causation of this disease.
Heredity, physical and metabolic activity, stress, endocrine
function and various environmental factors may interact
significantly with diet.

Experimental evidence of animals showing the influence

of stress on atherosclerosis are few, and often conflicting.

Sellers and You (1956) showed a positive effect of cold-

83

stress in inducing changes in coronary arteries of rats.
A similar result was reported by Schoenfeld (1960) in rats
following prolonged swimming in cold water.

Uhley t 22, (1957), on the other hand, reported no

definite relationship between the behavior of his chicks
(aggressive and passive groups) and the development of
arterial disease. However, food intake, weight gain, and
serum cholesterol levels of the aggressive and passive
groups were not equal.

Cholesterol-fed cockerels, when subjected to continued
anxiety by repeated electric shocks, gave no indication of
weight gain, blood lipid level change, or the development
of atherosclerosis (Joyner 22_22,, 1961). Gunn 22_22,
(1960) reported that rabbits fed on cholesterol excess diet,
and receiving chronic electric stimulation of their dien-
cephalon in the hypothalamic region, exhibited a greater
degree of aortic and coronary atherosclerosis than those not
receiving hypothalamic stimulation. They concluded that
central nervous system mechanism does exist which, under
certain circumstances, is capable of significantly influen-
cing arterial atherosclerosis.

The development of atherosclerosis is a silent phenom-

enon. The disease presents itself only when arteries become

84

incapacitated and complications are superimposed. For this
reason it is important to recognize the sub—clinical or the
deve10pmental stage of the disease. This, however, itself
raises a problem since direct examination of large arteries
22_y2y2_is not feasible.

Clinical studies on the role of psychological stresses
in atherosclerosis are also difficult to carry out. The
main reason for the difficulty lies in the lack of precise
measure by which to characterize psychological stress, and
to adapt such stress evaluation to the reaction of a given
individual (Katz 22_22,, 1959).

The blood of mammals and birds has been recognized as
a sensitive indicator of their internal conditions. The
liquid menstruum circulates in a closed circulatory system,
carrying with it the erythrocytes, the leucocytes, and
platelets or thrombocytes. This menstruum also carries a
mixture of electrolytes, amino acids, enzymes, hormones,
non-cellular proteins and other basic organic and inorganic
materials. The concentration of various proteins in the
circulation represents the balance between filtration into
the capillaries and return from the tissue spaces by way of
the lymphatic channel.

In a healthy adult of a given species, the concentration

85

of various serum proteins remains surprisingly constant.
Repeated electrophoretic analyses show patterns in which

not only the size of each major boundary, but also its shape,
remains constant. Hence, the concentration of minor com-
ponents within a major group must retain their ratios to
each other. This consistency of an individual's pattern

has been suggested to be under genetic control.

The determination of plasma protein levels as an aid in
diagnosis of diseases has such theoretical possibilities
that a vast amount of work has been expended on it (Ehrman-
traut, 1958). Because of the size of the liver and its
importance in protein metabolism, liver diseases change the
plasma protein markedly.

Electrophoresis is used for determining the isoelectric
point of proteins and also for the fractionation of protein
mixtures. It is customarily done in veronal buffer solution
at pH 8.6, at which the anodic migration is more pronounced.
Serum albumin, whose isoelectric point is near pH 4.6, moves
with the greatest velocity, the d-, B-, and y-globulins
with decreasing velocities. Quantitative evaluation of
these fractions, with subsequent increase or decrease of one
or more protein components of the serum, constitute the

"stress" pattern described by Dunn and Pears (1961).

86

According to Selye and Heuser (1956) the mechanism of
mammalian reaction to non-specific stress is as follows:

1. The stimulation is conveyed to the hypothalamus
Which causes the anterior pituitary to produce adrenocor-
ticotropic hormone (ACTH).

2. ACTH reaches the adrenal cortex and thereby releases
hormones which stimulate various organs, including gastric
glands of the stomach.

3. Pepsin and hydrochloric acid produced in the stomach
attack its contents and/or the stomach lining. Continuation
of this condition leads to gastric ulcers and malfunction of
both the liver and the kidney.

This study was undertaken in an attempt to evaluate
some environmental influence on the development of diet-
induced atherosclerosis in Coturnix quail (Coturnix cotugnix
ja2onica). It was hoped that these birds, being gregarious
in nature, would be suitable subjects for the study of the
effects of social isolation. The electrophoretic pattern
of the serum protein was analyzed in every case as a meas-
ure of physiological change, and it was hoped to provide
some diagnostic measure of the developmental stage of the

disease.

MATERIALS AND METHODS

Acqgisition and han22inggof chicks

Fertile Japanese Quail eggs, selected randomly from the
stock at the Michigan State University Experimental Poultry
Farm, were incubated in a "Jamesway 262" forced-draft type
incubator. The chicks hatched on the eighteenth day of
incubation, when they were removed from the incubator and
placed in "Jamesway" backwarmer type of starting batteries.
The chicks were reared on paper for the first seven days,
with the heating temperature adjusted to 95° F. At this
time, the paper was removed and the chicks were held in the
battery until they were two weeks old. During this period,
all the chicks were kept on standard chick starter ration.
The birds were then marked with leg bands, individually
weighed, and randomly divided into 36 groups (18 experi-
mental and 18 control). The final distribution of groups
was as follows: six groups each having 15 birds; six groups
each having five birds; and 24 groups each having a single

bird.

Housing

Each group finally was transfered to a partitioned pen

occupying one square foot of floor space. This resulted in

88

placing birds at concentrations of fifteen, five, and one
bird per square foot.

The pen available to house the quail had twelve com-
partments, six on each side, with the heating unit placed
at the center. This heating unit was raised 5.5 inches
above the wire mesh floor. Four compartments on either side
of the heating unit, excepting the top and the bottom
shelves, were partitioned off using plywood. This pre-
vented the birds, especially those that were individually
isolated, from seeing other birds kept in adjacent pens.
The finished pens measured 12 inches by 12 inches and con-
tained 144 square inches of floor space per pen. A common
water trough, with a partition made at the center, was used
for all four corner pens on each shelf. This partition
prevented the birds from mixing and also proved easier for
cleaning the trough when desired. The troughs on the outer

two sides of the pen were used as feeders for all birds.

Management
The experimental groups were kept on one side of the
I
heating unit, while the controls were keptcnithe other side.
Control groups were fed on a standard Japanese Quail Breeder

ration, developed at the Michigan State University Experi-

mental Poultry Farm (Table l). The experimental groups

89

Table 1. Japanese Quail Breeder Ration

 

Ground yellow corn . . . . . . . 38.25
Soybean oilmeal dehulled 50% . . . . 37.00
Alfalfa meal 17% . . . . . . . . . . 5.00
Dried whey . . . . . . . . . . . . 2.50
Meat and bone scraps 50% . . . . . . 2.50
Fish meal (Menhaden 60%) . . . . . . 2.50
Ground limestone . . . . . . . . . . 5.00
Dicalcium phosphate . . . . . . . . . 1.50
Salt (iodized) . . . . . . . . . . . 0.50
Vitamin premix (NOPCOSOL M-4) . . . . 0.25
Fat . . . . . . . . . . . . . . . . . 5.00

100.00

 

received the same ration except that their ration was for-
tified by adding 1 per cent of Nutritional Cholesterol by
weight. In order to make the two rations exactly compar-
able with respect to their caloric value, the ration for
the control birds was mixed with cottonseed oil 1 per cent
by weight. Feed and water were kept before the various
groups at all times.

Heating units at all levels were initially adjusted to
950 F. before the birds were placed in the experimental
pens. This was decreased gradually at the rate of 5 de-
grees per week until the temperature reached 80 degrees F.
The air temperature inside the pen ranged from 10 to 12
degrees below that recorded on the heater thermometer. The

temperature inside the room ranged from 40 to 70 degrees F.

during this experiment.

90

All groups received continuous lights on the 24-hour
basis which provided 15-20 foot candles of light at the
level of the feed and a minimum of 12 foot candles of light
in all parts of the pen. The experimental period lasted
from six to ten weeks. All the birds at the start of the
experiment were 15 days old. This was the experimental

design used in inducing atherosclerosis.

Handling of birds

Birds were little disturbed during the entire experi-
mental period. The pen was set in one corner of the room
which prevented the birds, at least on two sides, from
seeing other experimental birds housed elsewhere in the
same room. The remaining two sides were covered with thick
brown paper from outside, obstructing the bird's view and
preventing them from seeing other birds or the attendants
working in the room. Food and water were checked every day,
and the cleaning of the water troughs was done once or twice

a week as needed.

Collection of mategials
As initially planned, the first batch of birds was
ready to sacrifice at the end of six weeks. This included

one group of fifteen birds, one group of five birds, and

91

four groups of isolated birds. Birds were taken in this
fashion both from the experimental and the control groups.
The second and the third batches were ready at the end of
the eighth and tenth week respectively.

Birds were individually weighed and sacrificed by de-
capitation. Blood samples of 2-3 cc. were collected in
separate, sterilized centrifuge tubes, marked individually
for each bird. Samples were allowed to clot without agita-
tion and then centrifuged for 10 minutes at 1,500 rpm,
taking care to prevent haemolysis and subsequent foiling of
the whole sample. The serum thus separated from the blood
clot was finally transfered to small glass tubes marked
individually for each specimen.

An electrophoretic run of the sample was made as soon
as possible in order to avoid changes in the pattern due to
storage. Storage conditions can produce changes in electro-
phoretic pattern but the agreement among investigators is
equivocal. Meisner and Hickman (1962) reported that freez-
ing does not influence the electrophoretic pattern. How-
ever, they experienced difficulties with samples that had
been frozen.

After the sample was taken, a mid-ventral incision was

made, each bird eviscerated, and the sex and degree of

92

maturity were noted. Both the adrenal glands, situated
anterior to the testis or the ovary, were then exposed,
carefully detached from the surrounding tissue, and weighed
separately on a light precision balance. All the weights
were recorded to the nearest tenth of a milligram.

After these parameters were recorded, the heart and the
big blood vessels were exposed. These were carefully exam-
ined for evidence of atherosclerotic lesions. Portions of
suspected tissue were preserved for histopathological exam-
ination in a mixture of: commercial formalin five parts, 95
per cent ethyl alcohol 25 parts, glacial acetic acid one
part, and distilled water 20 parts (FAA). The preserved
tissues were later sectioned and examined under a compound

microscope.

Electrophggetic method

Fractionation of samples of the serum was performed
with Beckman Model R-101 Microzone Cell, using Gelman's
sepraphore III cellulose polyacetate electrophoresis support
membrane (2.5 X 12 cm). This membrane has uniform pore size
and distribution and has several advantages over other media
such as filter paper, starch, or agar. Some of these advan-
tages are: application to small samples (0.25 ml), minimal

absorption of proteins to this material, thereby eliminating

93

the tailing of fractions common to paper electrophoresis;
short running time; and ability to resolve samples into more
fractions with reasonable clarity. Also, the transparency
and the tensile strength of cellulose acetate after it has
been cleared with ethanol-acetic acid make this medium ideal
for scanning.

Barbital buffer (veronal) having a pH of 8.6 and ionic
strength of 0.075 was used both for soaking the strips and
filling up the Microzone cell. It consists of 1.66 gm bar-
bituric acid (crystals), 12.76 gm.of sodium diethyl barbitone
(powder), and sufficient water to make one liter. Storage
of the buffer solution was avoided as it is attacked by
microorganisms altering the conductivity of the solution.
Some authors recommend the addition of sufficient merthio-
late to obtain a concentration of 1 : 100,000, to prevent
spoilage. Sodium azide is also employed for this purpose.

The sepraphore membrane was soaked in the buffer by
carefully floating it on the surface of the buffer solution.
TPhis allows the membrane to wet uniformly, from the bottom,
53inking into the buffer quickly as wetting progresses. At
leeast an hour was allowed for soaking. This produces a col-
l<>idal change, bringing the membrane back to its original

gesl structure. Insufficient soaking may result in the

94

streaking or smearing of seprations.

While membrane was being soaked, the Microzone cell was
filled by tilting the fluid-leveling siphon to a horizontal
position and pouring buffer into one end of the siphon tube.
This was done until all the air bubbles were eliminated
from inside the tube. The siphon was then slowly released
until it returned back to its original position. Buffer
was poured in only one compartment which, in turn, was
automatically transfered to the other side by the siphon
tube. The cell was filled to a point between the two lines
marked "FLUID LEVEL." Enough time was given for the buffer
to equilibrate in both the compartments. If the buffer
level is not equal in both the compartments, capillary
action will take place through the membrane pores. Such
flowage will smear and distort the fractions and the strip

will be ruined.

Mounting the mem22ane on the bridge

After the membrane was thoroughly soaked in the buffer
solution, it was placed between two blotters using a cover
glass forceps. The membrane should never be touched with
bare hands, dry or wet. Blotters should remove only the
shining excess surface fluid. All operations done with the

membrane uncovered should be done as quickly as possible.

95

The strip was then quickly placed over the membrane
mount and secured properly by means of movable tensioning
membrane mount (Beckman Tech. Bull., RM—IM, 1963). The
bridge, properly mounted with the membrane, was then placed
in the cell, taking care not to splash any buffer onto the

membrane when the siphon flipped up.

Sample application

This process should be done as quickly as possible to
avoid unnecessary evaporation from the membrane surface
while the upper lid is off and to minimize diffusion of
samples in the membrane before current is applied. Samples
were applied using a sample applicator, taking all the pre-
cautions mentioned in Beckman Bulletin Manual (22, 222,).
The tip of the applicator was immediately ringed after each
application withaistrong jet of distilled water from a wash
bottle and dried by gentle touch of blotters. This precau—
tion was necessary to avoid mixing of one sample with an-
other. Eight samples were run at a time.

Power to the cell was supplied at constant voltage (250
v.) using a Spinco Model RD-2 Duostat. The run was made
for 20 minutes with a starting current of 3.5 ma to 5.8 ma,
and an ending current of 4.5 to 9.6 ma. When the ending

current went over 9.6 ma, the whole run was discarded.

96
Staining, c2eap2ng, a22,mounting of
222223 in the p2astic enve2ope

Various dyes may be used to locate the protein fractions
on the strip. A solution of Ponceau "S" described by Smith
(1960) was the stain of choice in this investigation. It
consists of 0.2 per cent Ponceau "S" in 3 per cent trichlor-
acetic acid. The uptake of this dyd, according to the above
named investigator, is quantitative to the amount of protein
on the strip.

At the end of the time allowed for the separation, the
power was turned off, and the membrane removed. The wet
strip was then laid over the surface of the stain, avoiding
air bubbles trapped underneath and allowing the stain to
soak into the strip from below. Seven to nine minutes was
allowed in each case for staining. The excess stain was
removed by gentle agitation for 3 minutes in each of the
three trays containing 5 per cent glacial acetic acid. It
is important to remove all background stain before quanti-
tation procedures are employed.

The membrane, after its background was completely white,
was laid over 95 per cent ethyl alcohol for dehydration for
about one minute. The strip was then transfered to another

tray containing 10 per cent glacial acetic acid in 95 per

97

cent ethanol (10 cc of glacial acetic acid and 90 cc of 95
per cent ethanol). The membrane was left in this solution
for approximately 30 seconds and finally spread over a clear
glass plate, taking precautions not to trap any air bubble.
Excess fluid was removed by tilting the glass plate and
squeezing the membrane gently and evenly by the smooth edge
of the plastic strip holder. The glass plate with the mem-
brane was then dried in a pre-heated oven (100 to 110 degrees
C.) until all the smell of acetic acid was gone. The mem-
brane was then carefully peeled off the plate and slipped
inside the plastic strip holder for storage and later

scanning.

Ey§2uation of the sepagated seggmgppoteins

The strips were scanned in a Spinco Analytrol Model RD
using R-102 Microzone Scanning Attachment. The analytrol
draws the curves for each component and the integrator
automatically indicates the exact area under each curve.
Both these functions are performed at the same time in a
single step.

While scanning cellulose acetate strips stained with
Ponceau "S" stain, a "B-2" cam or so-called "optical den-
sity cam" was used. The slit width of 0.5 mm, and a height

of 5 mm was used throughout the study. The original 500 mu

98

front interference filter used for scanning paper strips
was replaced by 520 mu interference filter. Strips were
inserted in the carriage by properly fixing them inside the
strip holder plastic covers. These transparent covers did
not interfere with the scanning process and ensured safety
of the membrane against the drive wheel. All strips were
scanned on an approximately equal distance between the cen-
ters of the first and last peaks on the chart. This was
obtained by adjusting the "SPEED CONTROL" knob at 45 in
every case. The percentage of each individual component for
statistical analysis was calculated as described in Beckman

Bulletin RIM-5.

RESULTS

A total of 160 Coturnix quail (75 males and 85 females)
used in this investigation were individually examined.

These specimens were randomly selected and are presumed to
be representative of the population. Also, the birds thrived
well under the entire experimental period. Only three
casualties were observed: one, a single isolated bird, died
due to accidental entanglement in the wire mesh of the cage;
and two probably were victims of fighting. The first group
was started again with a fresh specimen taken from the re-
serve. The latter two birds, however, which died near the
end of the experiment and although replaced by fresh birds
for the rest of the period, were treated exactly like other
specimens. Their data did not show any difference from that
of the other birds of their own group and are therefore
included in the final analysis.

A heterogeneous group of 16 birds were housed separately
in one complete shelf of the pen, without any space limita-
tion (875 square inches of space). These birds were kept
throughout the experiment on the basic Japanese Quail Breeder

Ration (page 89). The data obtained from these birds are

100

assumed to be normal for the species and therefore are

used for all comparisons in this investigation.

Body weight and adrenal weight

The mean body weights of the three population densities
are shown in Table 2. The mean body weight of the birds
kept in the group of 15 and isolated for 10 weeks was sig-
nificantly smaller than the mean body weight of the normal
birds (P = .05). This difference, however, was not con-
sistent with other three stages of isolation (six, eight,
and ten weeks). At the start of the experiment (two weeks
of age), the mean weight of the females was not significantly
different from the mean weight of the males. However, at
six, eight, and ten weeks of isolation the mean weight of
the females was significantly greater than the mean weight
of the males (P = .05).

Table 2 further shows the mean weight of the adrenal
glands (mg) calculated for each 100 gram of quail's body
weight. The mean weight of the adrenals in the group of 15
and in individually caged birds at eight and ten weeks of
isolation was always higher than the mean weight of the
adrenals of the normal birds. These differences are sta-

tistically significant at the five per cent level. The

101

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102

'birds caged in the group of five, however, did not show any
difference; neither in their body weights nor in the weights

of their adrenal glands.
Electrophoretic pattern of the serum protein

2222222birds

Serum samples from each specimen were run for electro-
phoretic pattern determination as described earlier. In
most cases, at least seven distinct fractions were obtained.

The two sub-fractions of the d-globulin (d and 02), in some

1
cases, however, did not separate with distinct clarity. In
the final analysis, therefore, these two sub-fractions were
lumped together. Few specimens, on the other hand, revealed
a distinct band of a fast moving protein fraction (pre-
albumin). This was observed both in the normal and in the
treated birds. The quantity of this fraction, however, was
so small that it was not considered for statistical analysis.
A striking difference in the gamma globulin fraction of
the male and the female bird was also noticeable in this
investigation. This was found consistent throughout the
experiment both in the normal and in the treated birds. In
the normal female birds, this fraction represented 8.50 I

0.54 per cent of the total serum protein as against 4.44 i

103

1.11 per cent of the serum proteins of the males. This
difference is significantly greater at the five per cent
level. The curve, as appeared on the graph after scanning
(Figure l), is traced and shown in Figure 2.

A greater quantity of this fraction in the females was
balanced by a reduced quantity of their albumin fraction.
Albumin was 36.46 I 0.81 per cent of the total serum pro-
teins in the females as compared to 46.93 i 2.48 per cent
of the males. This difference, again, is significant
statistically (P = .05). Table 3 shows the difference in
the electrophoretic pattern of the serum proteins of the
normal male and female Coturnix quail. Other globulin
fractions, however, did not show marked sex differences.
The last column of Table 3 represents the percentage frac-
tions of total serum proteins of male and female birds com-
bined. These pooled values are used for all other compari—

sons in this investigation.

.222ds kept on cho2ester22-mix diet

The electrophoretic pattern of the serum protein of
birds kept on cholesterol-mix.diet shows a different picture
from that of normal birds. A slight decrease in the albumin
and in the alpha-globulin fraction was almost consistent.

Differences in these fractions, however, were not

104

Figure 1. Analytrol showing a scanned graph.

 

 

 

 

106

Figure 2. Electrophoretic pattern of the serum of a
normal female Coturnix quail (Coturnix coturnix ja2onica).
Notice the sharp peak in the region of Vi globulin.

Figure 3. Electrophoretic pattern of the serum of a
normal male Coturnix quail (Coturnix coturnix ja2onica).

 

 

 

 

 

 

 

 

 

Alb.

 

Alb.

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108

 

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109

sstatistically significant. The most striking difference
cibserved was in the beta-globulin fraction. There was a
(zonsistent increase in this fraction at the density of 15
lairds per cage and in birds caged singly at all stages of
j_so1ation (six, eight, and ten weeks). The only group that
(deviated from this general trend was the individual birds
:Lsolated for eight weeks. All these differences are sig-
riificant statistically at the five per cent level. A general
'trend toward decline was also observed in the gamma—globulin

fraction. These differences are shown in Table 4.

JBirds kept on non-cholesterol diet

 

The electrophoretic pattern of the serum protein of
loirds kept on cholesterol-free diet (Table 5) showed little
car no difference from that of the normal birds (Table 3).
'The only difference noticed was in the beta and gamma
sylobulin fractions which showed a tendency toward decline.
This reduction of the gamma-globulin was significant at
least in two groups of birds at the population density of
five and fifteen and at six and eight weeks of isolation
(P = .05). The significant decrease in the beta-globulin
was also found in the same group of five birds isolated for
eight weeks, and one group of birds caged singly and iso-

lated for ten weeks (P = .05). A striking difference,

110

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111

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112

however, was evident when the electrophoretic pattern of the
whole group was compared with the electrophoretic pattern of
the serum protein of the birds kept on cholesterol—mix diet
(Table 4). On the whole, the general increase of the beta-
globulin fraction in the cholesterol-fed birds was repre-
sented by a consistent decline in the beta-globulin fraction
of birds kept on non-cholesterol diet.
Incidence of disease in the
expe2222nta2;and contro2¥bird2

The birds subjected to group isolation showed striking
differences in atherogenesis. Diet-induced atherosclerosis
was significantly enhanced in the birds subjected to group
isolation, especially at the population density of 15 and in-
socially isolated birds caged singly. Out of 45 birds in
the three experimental groups (kept on cholesterol-mix diet
at the density of 15) 18 developed the disease; two at six,
five at eight, and eleven at ten weeks of isolation. Among
the socially isolated birds, six out of twelve developed the
disease within eight and ten weeks. One of these birds,
however, belonged to socially isolated group kept on choles-
terol-free diet. At the population density of five, kept on
cholesterol-mix diet, only one developed the lesions at ten

weeks isolation. These differences are significant

113

statistically at 0.1 per cent level (chi-square = 59.79 with
17 degrees of freedom). Sex, however, did not seem to
effect atherogenesis in this investigation. Out of 45 birds
developing arterial lesions, 15 were males and 10 females.
This difference is not significant statistically.

Figures 4, 5, and 6 show the formation of atheromatous
plaques in the aorta of Coturnix quail. Three distinct
stages were recognized which were distinguished on gross,
naked eye examination.

a) Lipid deposit: Superficial yellow or greyish-yellow
lesions on the intima visible without a microscope.

b) Fibrous plaques: Circumscribed thickening of the
intima producing a firm, grey or glistening prominence.

c) Atheroma: A plaque in which fatty softening pre-
dominated. In the stained sections, the fatty material was
dissolved by the preservative (FAA) and represented empty

vacuoles.

Behavioralidifferences

In addition to the above parametric observations,
interesting changes in the behavior of birds isolated singly
were noticed. For the first two weeks, they produced more
noises than other birds caged in the group of 15 and five.

Their calls invariably were replied to by other birds

114

Figures 4 and 5. Formation of atherosclerotic plaque in
the lumen of aorta. Top picture (Fig. 4) shows the cross
section of a normal aorta. Bottom picture (Fig. 5) shows
a big atheromatous plaque in the lumen. Deposits within
the intima are hardened and the lumen has been narrowed
considerably. Hematoxylin-eosin. 40 X.

 

ll» I.
D

 

 

 

116

Figure 6. An advanced case of atheromatous plaque in the
aorta. Note the endothelial lining and a much thickened
intima infiltered with lipid deposits. Hematoxylin-eosin.
290 X.

1. Lumen with blood cells

2. Endothelial lining

3. Thickened intima

4. Lipid vacuoles

5. Media or the muscular layer

 

 

118

isolated singly. Later, these birds developed neurotic
behavior marked by an exceedingly strong tendency to be

over active, interrupted only by short intervals. They
scratched in their feeding troughs and watering bottles with
an untiring persistence, flapping their wings and scattering
the food without actually eating. When I cleaned their pens
or refilled the water bottles, they actively resisted and
violently attacked with extreme excitability. The birds
kept in the group of 15, on the other hand, were more timid

and either crouched or tried to escape while being handled.

DISCUSSION

Body weight and adrena2 we2ght

The well established pituitary-adrenal relationship
of mammals described elseWhere (page 86) is not so well
defined in birds. Evidence from recent studies indicates a
relative autonomy of the adrenocortical tissue of birds
(Flickinger, 1959). Nonetheless, in this experiment, the
adrenals of Coturnix quail kept at high density levels have
been heavier than the adrenals of birds kept at low density.
Siegel (1959; 1960), in the case of his chicks, attributed
this difference to adrenocortical hyperplasia.

Social order, especially among the cocks, seemed to be
more important in very crowded pens. Hens were usually
subordinate to cocks. This was evident from direct obser-
vation. These birds were poorly feathered and their plumage
was frayed and dirty. However, they did not show a greater
number of injuries and their health appeared to be equivalent
to that of the birds in less dense populations.

Failure to show significant difference either in the
body weight, or in the weights of their adrenal glands at
six weeks of isolation can be explained by the fact that the

birds had not yet reached sexual maturity. The social

120

hierarchy in the males probably becomes more rigidly estab—
lished only after they have attained sexual maturity (about
8 weeks). It was because of this sexual maturity and sub-

sequent social hierarchy that a significant correlation of

adrenal weights was established in the latter two groups at
eight and ten weeks of isolation.

The occurrence of even larger adrenals in the socially
isolated birds suggests that smell, sound, or even sight
reactions as a whole may be important in eliciting adrenal
responses. This was evident from the changed behavior of
these birds which were greatly excited at the call of other
birds kept in different parts of the pen. These facts,
rather than the social interactions between the members of
the group, seem to be the possible explanation for larger
adrenals in the birds caged singly as compared to the birds
kept at the density of 15.

Failure to obtain a consistent difference in the body
weights of the Coturnix quail kept at higher density, or
even in socially isolated ones, suggests their remarkable
adjustment to crowding. In the light of this observation,
lipid depletion in the cortical tissue, or even direct com-
parison of corticosteron in the circulating blood would

give a better estimate of a “stress" situation in these birds.

121

A significant increase in the body weight of females at
all density levels, and at all stages of isolation, however,
was expected. This occurred with Coturnix quail housed
under various environmental conditions (Stanford, 1957;

Wilson 22_22,, 1961).

The electrophoretic pattggn of serum protein

From the electrophoretic analysis of the various serum
proteins of the normal and experimental birds it is obvious
that some variation in the distribution of the blood pro-
teins occurs from individual to individual. This variation
appears as differences in mobilities, in the number and
relative proportions of components, and in the failure of
some sera to show good resolution of various fractions.

In many of the sera studied it was rather difficult to
ascertain the amount of particular globulin present, pri-
marily because they were not sufficiently separated. In
other words, differences in their mobility in an electrical
field were too small for good resolution. This was partic-
ularly true in the case of alpha and gamma globulins, in
which evidences of the presence of these proteins were
sometimes seen in the form of a sharp peak superimposed on

a mass of some other protein fraction. No explanation for

122

this difficulty could be given at present as within the same
electrophoretic run samples which did separate well and

those which did not were found. Due to poor resolution, the

areas of fractions d - and d -, and 51- and 82-, were lumped to-

1 2

gether for each specimen to allow a better comparison of the
data. These groupings may have masked some significant
changes within these fractions, but no alternative was
available.

Certain specimens, on the other hand, showed the pres-
ence of protein which migrated with a mobility somewhat
higher than that of albumin. These faster moving components
(pre-albumin) did not seem to occur constantly, but appeared
in various serum samples in a random fashion, in the males
and females taken from control as well as experimental
groups. No biological activity can yet be attributed to
this protein, although binding of thyroxine by a pre-albumin
has been demonstrated in man (Tata, 1959). Random presence
of pre-albunin in very small quantity has also been reported
in the plasma of other avian species like white Leghorn,
bronze turkeys, ring-necked pheasants, pigeons, and muscovy
ducks (Deutsch and Goodloe, 1945). There are no studies,
however, to my knowledge, on electrophoresis of serum pro-

tein of Coturnix quail, other than the one presented here.

123

Also, it seems to present the first report on the effects
of social isolation in experimental atherogenesis in this
animal.

Gamma—globulin in the Coturnix quail, like human gamma-
globulin, migrated in a peculiar way. Because of electro-
endosmosis and other unexplained phenomena at the end of
electrophoresis, the band stretched from the origin toward
the cathode end, forming a wide band. A second peak, how-
ever, consistently appeared in the females in the region of
yl. Differences in this particular globulin were so pro-
nounced that a mere visual inspection of the pattern was
enough to predict sex with almost 100 per cent accuracy.

Antibodies in the sera usually migrate with a mobility
near that of gamma-globulin, but no electrophoretic differ-
ences can be detected between physiologically occurring
gamma-globulin and that containing immune bodies. It has
been suggested, however, that all gamma-globulins are anti-
bodies (Anker, 1960). This basis is supported by the fact
that in animals raised under sterile conditions the gamma-
globulin concentration is almost negligible. In human
beings, hyperfunction of the adrenal gland, or those treated
with ACTH or cortisone, have low levels of gamma—globulin

possibly due to lysis of lymphatic tissue (Vaughan t al.,

 

124

1951). If this is true also for birds, then it might explain
the higher gamma-globulin fraction in the normal Coturnix
quail kept in unlimited space free from stress and reduction
(although not consistent) in birds kept at the density of 15
or caged singly due to crowding or social stress.

The most striking difference, as expected, however, was
found in the beta-globulin fractions of normal and experi-
mental birds kept on cholesterol-mix diet at the density of
15 or caged singly. These globulins often were concerned
with the lipid present in the serum, as indicated by the
movement of turbidity with these fractions. This was further
confirmed by pre-staining the serum lipoproteins with Sudan
Black-B and then comparing the blue-band zones of lipo-
proteins with other protein fractions stained in Ponceau "S."
In the case of Coturnix quail studied, this turbidity mi-
grated on the fast side of globulin complex and seemed to
be associated predominantly with the beta-globulin and less
so with alpha and gamma globulins. In humans, beta-lipo-
proteins form approximately 5 per cent of the plasma proteins
and contain about 70 per cent of the plasma lipids (Hauro-
witz, 1963). In the present study, quantitative analysis of
various lipoproteins was not done, but the stained patterns

in Sudan Black indicated that at least 60-70 per cent of

125

the lipids were bound up with beta-globulins.

In the light of the above observations it is apparent
that environmental factors, which might alter the psycho-
logical state of quail, can influence the development of
dietary-induced atherogenesis. The high incidence of dis-
ease in birds kept at a population density of 15 per square
foot and in socially isolated birds kept on equal floor
space, accompanied by a significant increase in their beta-
globulin fraction could alone justify the above observation.
However, the mechanism underlying the effect of such en-
vironmental influence on atherogenesis is not clear.
Decreased physical activity in the birds kept at higher
density level have to be considered. But equal or even
greater susceptibility to the disease in over active, singly
caged birds makes physical activity 222_§2_an unlikely
explanation. It is possible that psychological stress in
these birds might have influenced the cholesterol metabolism
by release of cortisone which is known to elevate the blood
cholesterol levels.

Further study might be directed toward isolating the
types of environmental changes which can influence athero-

genesis and the exact mechanism involved.

SUMMARY

In view of previous findings, it seems that crowding,
beyond certain limits, does affect the adrenal function.
In Japanese quail, kept in the group of 15 per square foot
of space, adrenals were significantly heavier than the
adrenals of birds kept in identical situation in the group
of five. Even larger adrenals were found in socially iso-
lated birds caged singly. Stimulation of adrenal glands
correlated well with the amount of social interaction, or
intolerance of individuals towards others of their own group,
at higher population.levels (15 birds per cage). This was
evident by direct observation and also by the amount of
injuries present and the loss of their body plumage. Social
hierarchy in males developed only after they had attained
sexual maturity (about 8 weeks). After this stage, the
adrenals of birds kept in the group of fifteen were signif-
icantly heavier than the adrenals of birds kept five together.

Olfactory, acoustic, and even visual stimuli seem to
evoke adrenal responses in socially isolated animals. This
was supposed to be the only reason for heavier adrenals
found in Coturnix quail caged singly in this investigation.

The exact neural mechanism, and nature of the controlling

127

process or processes involved, is still unknown.

Coturnix quail are endowed with a remarkable power for
adjustment to crowding. Despite significant changes in the
gross weight of adrenals, no significant difference was
found in their body weights.

A highly unnatural situation in the form of social
isolation or intense crowding imposed on Coturnix quail in
this investigation augmented experimentally induced athero-
genesis. Incidence of the disease was significantly en-
hanced in birds fed on cholesterol-mix diet and kept either
in the group of 15 or caged singly in complete social iso-
lation. During the entire experimental period, only one
bird out of 15, kept on atherogenic diet and not subjected
to crowding or to social isolation, developed the disease.
This suggests that, besides diet, there are multiple inter-
acting factors to influence atherogenesis. Isolation of
these environmental factors, and the exact mechanism in-
volved, is the subject for further research.

The electrophoretic pattern of serum protein seems
promising as an index to an individual's physiological
state. Increased amounts of beta-globulin components in
the birds kept on the atherogenic diet and subjected to

crowding or to social isolation seem to be a reliable

128

indication of the altered nutritional-metabolic cholesterol-
lipid-lipoprotein state of an individual. A significant
increase in this component, therefore, could be taken as a

reliable guide for the developmental stage of the disease.

LITERATURE CITED

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1963. Social environment, emotionality, and alloxan
diabetes in the rat. Psychosom. Med., 25:60-68.

Ader, R. and E. W. Hahn.
1963. Effects of social environment on mortality to
whole body X-irradiation in the rat. Psychol.
Rept., 13:211-215.

Ader, R. and S. B. Friedman.

1964. Social factors affecting emotionality and resist-
ance to disease in animals: IV. Differential
housing, emotionality, and walker 256 carcino-
sarcoma in the rat. Psychol. Rept., 15:535-541.

Andervont, H. B.
1944. Influence of environment on mammary cancer in

mice. J. Nat. Cancer Inst., 4:579-581.

Anker, H. S.
1960. The biosynthesis of plasma proteins. 22_The
plasma proteins, ed. by F. W. Putnam, Academic
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Anonymous.
1949. Bird house renovation at Philadelphia. Park and
Recreation, 32:51.

Anonymous.
1961. Tropical rain forest San Diego Zoo. International
Zoo Yearbook, II:67-68.

Anonymous.
1961a. Dimensions of moats and ditches at Detroit Zoo.
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Anonymous.
1962. Bird halls at Frankfurt Zoo. International Zoo
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Appelman, F. J.
1961. Feeding of zoo animals by the public. Interna-
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Beckman Manual.
Beckman model R paper electrophoresis system
instruction manual No. RIM - 5. Spinco Division
Stanford Industrial Park, Palo Alto, California.
60 p.

Beckman Model.
1963. Model R-lOl microzone electrophoresis cell pre-
liminary instruction manual. Tech. Bull., RM-IM-Z.
Spinco Division, Beckman Instruments Inc., Stanford
Industrial Park, Palo Alto, California. 38 p.

Bergh, W. V. D.
1960. The new ape house at Antwerp Zoo. International
Zoo Yearbook, I:7-11.

Berridge, W. S.
1932. Zoo animals. A & C Black, Ltd., London. 96 p.

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