A STUDY OF NITROGEN METABOLISM
WITH SPECIAL REFERENCE TO MINK.
James Edmund O l d f i e l d
A thesis submitted i n p a r t i a l f u l f i l m e n t
of the requirements f o r the degree of
Master of Science i n Agriculture.
IN THE DEPARTMENT
OF
ANIMAL HUSBANDRY
The University of B r i t i s h Columbia
August, 1949.
A STUDY OF NITROGEN METABOLISM WITH SPECIAL R2PERSNCE TO MINK.
by
James Edmund O l d f i e l d
ABSTRACT:
Experimental studies with mink at the University of
B r i t i s h Columbia had t h e i r o r i g i n with the a c q u i s i t i o n by the
University of a mink colony i n 1947. In September of that
year various l o c a l mink ranchers donated some 60 animals to
the University with a view to establishing an experimental
unit on which research might be c a r r i e d out. The ultimate -
object of such research was to be the formulation of d e f i n i t e
feeding standards f o r mink, such as are already available f o r
other species. This project was recognized as a long-term
proposition, and i n i t i a l experiments were designed to inves
tigate the protein requirements, of mink.
Preparatory to the experimental project, a survey of the
l i t e r a t u r e concerning general nitrogen metabolism,'and more
p a r t i c u l a r l y the concept of so-called "endogenous" nitrogen
metabolism was c a r r i e d out. * This survey constitutes the
opening portion of t h i s Thesis.
The actual experimental work undertaken was divided into
two phases:
1. Investigation of the endogenous nitrogen excretion
of mature animals maintained i n a f a s t i n g condition, or on
a nitrogen-free d i e t .
...ABSTRACT (2)
2 . Conventional nitrogen balance t r i a l s , involving the
establishment of nitrogen equilibrium at the lowest possible
l e v e l , using certain s p e c i f i e d sources of dietary protein.
The method followed involved the c o l l e c t i o n and analysis
of urine samples from adult animals maintained under d i f f e r e n t
stated conditions of n u t r i t i o n . Total nitrogen was determin
ed by the Kjeldahl-Gunning procedure, and creatinine content
was estimated by the F o l i n - J a f f e a l k a l i n e picrate reaction.
Various supplementary procedures were instigated to guard .
against possible interference by abnormal urinary constituents.
The results obtained would appear to have extensive imp
l i c a t i o n s regarding future investigations into the n u t r i t i v e
requirements of mink. F i r s t , at the expense of a great deal
of time and e f f o r t , equipment has been b u i l t which has proven
satisfactory for the laboratory Investigation of t h i s newly
domesticated animal. Second, the close c o r r e l a t i o n of actual
data with figures c i t e d i n the l i t e r a t u r e f o r other species
of s i m i l a r bodily dimensions suggests that the mink i s not
phys i o l o g i c a l l y abnormal, and that predictions as to i t s nut
r i t i v e behaviour may be made i n comparison with.other species
with reasonable accuracy. Third, the experiments dealing
with protein requirements suggest that considerable overfeeding
of proteins may be common prac t i c e , especially i n cases of
mere maintenance of mature animals. A very strong suggest
ion i s put forward f o r future studies into the b i o l o g i c a l
values of d i f f e r e n t native proteins f o r mink.
...ABSTRACT (3)
Detailed descriptions of a n a l y t i c a l procedures and ex
perimental equipment, and discussion of additional topics
regarding mink n u t r i t i o n are appended'to the main body of the
Thesis, i n the hope that they may serve as a useful reference
f o r future investigations along t h i s theme. These l a t t e r
include figures representative of time of passage and basal
metabolism; reference to the natural diet of the mink i n the
wild state; correlations between organ size and body weight
i n mature animals, and weight changes exhibited by growing
mink k i t s .
Approved T H . M . King) Frofessozyand
Head, Department of Animal Husbandry.
ACKNOWLEDGEMENT
The writer wishes to acknowledge h i s appreciation to
Professor H.M. King, Head of the Department of Animal Husband
ry f o r putting at his disposal the resources of the Department
and especially the University Mink Colony on which the exper
imental work contained herein was ca r r i e d out.
Special thanks are tendered Dr. A.J. Wood, Associate
Professor i n the Department of Animal Husbandry f o r the ben
e f i t 'of h i s teaching i n Animal Nutrition, his keen c r i t i c i s m ,
and his boundless encouragement and enthusiasm in'the conduct
of t h i s project.
The writer also wishes to acknowledge the co-operation
of various organizations interested i n the advancement of
science i n agr i c u l t u r e . A scholarship administered by the
A g r i c u l t u r a l I n s t i t u t e of Canada made i t f i n a n c i a l l y possible
f o r the writer to carry out t h i s program of research. A
grant from the B r i t i s h Columbia Ind u s t r i a l and S c i e n t i f i c
Research Council made possible the provision of the necessary
apparatus and supplies.
A STUDY OF NITROGEN METABOLISM WITH SPECIAL REFERENCE TO MINK.
TABLE OF CONTENTS PAGE
I INTRODUCTION 1 The general scope of nitrogen metabolism i n
vestigations and balance t r i a l s with a note re t h e i r p a r t i c u l a r a p p l i c a t i o n i n the case of mink.
I I LITERATURE SURVEY 3 General Nitrogen Metabolism 3 Amino Acid Metabolism 3 Enzymatic Action 5 Absorption 6 Functions of Nitrogenous Compounds i n the Tissues 8 S p e c i f i c Dynamic Action 11 Protein Storage 13 Mi c r o b i a l Action 15
Endogenous Nitrogen Metabolism 17 Theories of Endogenous Nitrogen Metabolism 17 Measurement of Endogenous Metabolism 19
The Significance of Nitrogen Balance 23 D e f i n i t i o n 25 Calculation of the State of Nitrogen Balance 23
N u t r i t i v e Value of Proteins 25
II I EXPERIMENTAL 31 Some Considerations Involved i n Planning a
N u t r i t i o n Experiment. Choice of F i e l d f o r Experimentation 33 Plan of Experiment 34 Method 35
Observations and Discussions 39 Endogenous Nitrogen Excretion 39, Nitrogen Balance Experiments 42 Creatinine Excretion ' 48
Summary 51
IV APPENDICES i
Preparation of Reagents and Laboratory Tech- i niques.
Animal Techniques v i
Additional Data Re Mink N u t r i t i o n x
A STUDY OF NITROGEN METABOLISM WITH SPECIAL REFERENCE TO MINK
In both plants and animals a substance i s contained which i s produced within the former and imported through the food to the l a t t e r . I t i s one of the most complicated substances ... very changeable i n compositi o n . I t i s unquestionably the most important of a l l known substances i n the organic kingdom. Without i t no l i f e appears possible on our planet. Through i t s means the chief phenomena of l i f e are produced.
- G. J . Mulder (1840)
INTRODUCTION
Possibly no' single phase of metabolism has been the sub
j e c t of so much controversy, or the object of more det a i l e d
investigation than has the metabolism of proteins and of n i t
rogenous compounds generally. From the earliest days of the
study of the science of n u t r i t i o n , investigators have believed
proteins to be endowed with some v i t a l property not attributed
to the other classes of nutrients. In the words of Rubner,
(1920), one of the pioneers i n t h i s f i e l d , "Protein contains
the magic of l i f e , ever newly created and then dying, a process
continuous since the advent of l i f e upon the earth." Among
the major foodstuffs, carbohydrates and fats contain b a s i c a l l y
only carbon, hydrogen and oxygen i n various proportions.
Proteins, i n addition to these elements, contain nitrogen and
usually sulphur, and quite commonly other inorganic elements
including phosphorous, i r o n and copper. Perhaps even more
important than the mere presence of these elements, however,
i s t h e i r organization into the complexity of form i n which
they are f i n a l l y used by the organism. Through t h e i r complicat
ed structure, therefore, as well as t h e i r diverse d i s t r i b u t i o n ,
- 2 -
("Protoplasm sans protein does not e x i s t " - C a h i l l (1944,a),
proteins o f f e r to the student of n u t r i t i o n a f i e l d which i s
at once absorbing and enlightening.
Not a l l of the nitrogenous constituents of the animal
body are proteins; however by v i r t u e of t h e i r vast quantita
t i v e s u p e r i o r i t y and t h e i r metabolic s i m i l a r i t y to the other
nitrogenous components, proteins are commonly assessed as a
v a l i d expression of nitrogen metabolism as a whole. In the
following pages a survey of the l i t e r a t u r e i s presented i n an
attempt to c l a s s i f y and explain, as f a r as i s presently
possible, the various phases of nitrogen metabolism as inves
tigated i n various animals and f i n a l l y an experiment i s des
cribed wherein some of these data are applied i n the s p e c i a l
case of the mink.
Since the time of Sanctorius of Padua, (Lusk, 1931a),
the value of so-called "balance t r i a l s " i n the in v e s t i g a t i o n
of n u t r i t i v e requirements of animals has been recognized.
Nitrogen balance, or the precise comparison of nitrogen i n
the ingesta and excreta of an animal, serves as ah i n d i c a t i o n
of the nitrogen metabolism of that animal. In addition, the
importance of nitrogen balance studies i s enhanced by the
f a c t that a supply of proteins or t h e i r components i s i n d i s
pensable to higher organisms as already mentioned (Jackson,
1945). A study of the theory of nitrogen metabolism comple
mented by an experimental nitrogen balance t r i a l i s p a r t i
c u l a r l y applicable i n the case of mink for several reasons.
The incomplete domestication of these animals causes them
to be extremely resentful of changes i n t h e i r environment,
necessitating rather c a reful d e f i n i t i o n of experimental con
d i t i o n s . In view of t h i s f a c t , i n v e s t i g a t i o n of the nitrogen
metabolism of mink which as t y p i c a l carnivores h a b i t u a l l y
consume diets r i c h i n meat and f i s h would seem a l o g i c a l
s t a r t i n g point i n the c a l c u l a t i o n of t h e i r complete n u t r i t i v e
requirements.
LITERATURE SURVEY
General Nitrogen Metabolism
The source of the great majority of the nitrogen meta
bolized by the animal body i s the dietary protein. Some food
materials, notably green vegetables and roots, contain apprec
iable quantities of free amino acids; however i n p r a c t i c a l
n u t r i t i o n i t i s doubtful whether these can be considered as
important sources of nitrogen. Demonstration of anaphylactic
shock i n sensitized animals i n cases of certain protein a l l e r
gies (Wilson, 1935), suggests the d i r e c t absorption of at
lea s t small traces of proteins from the digestive t r a c t .
This operation probably occurs i n minute quantities only,
however, and the b i o l o g i c a l value of such absorption i s doubt
f u l .
Amino Acid Metabolism
The c l a s s i c a l view of nitrogen metabolism involved the
hydrolytic breakdown of dietary protein into i t s constituent
amino acids followed by the recombination of these same amino
acids to y i e l d the proteins necessary to the animal body. I f ,
- 4
as i s often the case with "normal" diets f o r mature animals,
more protein i s supplied than i s necessary to f u l f i l the
several requirements f o r nitrogenous materials i n the body,
the excess i s degraded and i t s nitrogen eliminated mostly i n
the forms of urea, ammonia, or u r i c a c i d . (Baldwin, 1947a).
I t i s also immediately evident that as proteins are complex
conjugations of amino acids i n the higher animals at l e a s t
i t i s these amino acids which are the ultimate l i m i t i n g f a c
t o r i n the entire scheme of nitrogen metabolism. Obviously,
i t w i l l be impossible to b u i l d a protein within the animal
body i f one of i t s constituent amino acids i s unavailable,
therefore the necessary amino acids must eithe r be supplied
i n the d i e t or synthesized from other substances by the animal.
Preliminary studies on the biochemistry of proteins were
contingent on the development of suitable methods f o r the
i d e n t i f i c a t i o n and separation of amino acids. I t was d i s
covered quite early that proteins when boi l e d with strong
mineral acids would break down into mixtures of amino acids
but the i s o l a t i o n of these l a t t e r remained a perplexing prob
lem u n t i l the a p p l i c a tion of the low pressure f r a c t i o n a l
d i s t i l l a t i o n technique by Emil Fischer i n 1901. This method
involved the f r a c t i o n a l d i s t i l l a t i o n of the ethyl esters of
amino acids under conditions of very low pressure, and i t
resulted i n very rapid progress i n the knowledge of protein
structure. Even the adoption of such improved techniques
could not completely c l a r i f y the scheme of protein constitu
t i o n , however, due to the extreme complexity of the native
substances. That i s to say, although p a r t i a l fragmentation
of the protein molecules and i d e n t i f i c a t i o n of t h e i r compon
ents could be carried out, the remainders involved immense
technical d i f f i c u l t i e s and our knowledge on the subject i s
s t i l l f a r from complete.
Enzymatic Action
The l i b e r a t i o n of the i n d i v i d u a l amino acids from t h e i r
mother proteins takes place i n the i n t e s t i n a l t r a c t and i s
the culmination of many and varied enzymatic reactions. A
complete description of the mechanisms of these breakdown
processes i s a study i n i t s e l f and l i e s beyond the scope of
t h i s paper; however, a b r i e f resume i s necessary f o r contin
u i t y of the theme. Enzymatic action upon proteins i s hydro
l y t i c i n nature - that i s , causing a cleavage with the addi
t i o n of the elements of water at the point of cleavage.
(Mitchell, 1929a). One of the important features of such a
breakdown procedure i s that i t e n t a i l s an almost n e g l i g i b l e
loss of chemical energy thus r e s u l t i n g i n a high conservation
of energy i n the digested products. Hydrolysis proceeds
stepwise, r e s u l t i n g i n the successive formation of smaller
and smaller fragments of the mother protein accompanied at
each stage by the l i b e r a t i o n of c e r t a i n amino acids or amino
acid groups.
Digestion of dietary protein commences i n the stomach
through the action of a p r o t e o l y t i c enzyme, pepsin, i n the
strongly acid medium of the g a s t r i c j u i c e . Another stomach
enzyme, rennin, converts milk casein to paracasein which
- 6 -
forms an insoluble calcium s a l t (curd) thus retarding the
rate of passage of th i s food through the digestive t r a c t while
increasing the absorptive surfaces by distension. Pepsin,
of course, acts upon the protein of the c l o t i n i t s usual
manner. Action of these enzymes c a r r i e s the fragmentation
of the native proteins to the phases of metaproteins, pro
teoses and peptones. Following these i n i t i a l breakdown pro
cedures, the protein materials are transported into the i n
testine as a component of chyme, whereupon they are subjected
to more vigorous action by the pancreatic enzyme, trypsin,
and the i n t e s t i n a l enzymes, erepsin. The mode of action of
these l a t t e r i n common with that of pepsin i s characterized
by the formation of proteoses and peptones, however, the
l i b e r a t i o n of increased quantities of amino acids indicates
a somewhat d i f f e r e n t point of attack. Experiments conducted
by Frankel (1916) would seem to point to a complementary ac
ti o n by the various p r o t e o l y t i c enzymes - that i s , the hydro-
l y t i c effects of t r y p s i n and erepsin are more complete when
preceded by peptic digestion.
Absorption
Considerable doubt s t i l l e x ists as to whether proteins
are absorbed e n t i r e l y i n the amino acid form, however an
overwhelming mass of evidence points to t h i s method of ab
sorption as the normal procedure. The presence of hy d r o l y t i c
enzymes capable of cleaving the native proteins into t h e i r
constituent amino acids i n the digestive t r a c t ; the occurr-
ence of considerable quantities of free amino acids i n the
- 7 -
i n t e s t i n a l contents; and the apparently normal n u t r i t i v e con
d i t i o n of animals fed amino acids i n place of proteins a l l
lend weight to t h i s concept. Absorption of the end products
of protein digestion appears to be most active i n the duodenal
region where an extensive surface i s presented through the
involutions or v i l l i of the i n t e s t i n a l mucosa.
SCHEMA OF INTESTINAL VILLI (DOG)
... a f t e r Maximow and Bloom (1947)
I t w i l l be noticed from the diagram presented above that
two pathways are open to the products of protein digestion
absorbed through the v i l l i underlying the i n t e s t i n a l mucosa.
They may enter the blood c i r c u l a t o r y system d i r e c t l y through
the c a p i l l a r y network or i n d i r e c t l y by way of the l a c t e a l s ,
the lymph c i r c u l a t i o n and the jugular vein. I t i s generally
- 8 -
accepted that protein and carbohydrate products take the
former path and fat s the l a t t e r although the separation of
the nutrients does not appear complete and i s by no means •
thoroughly understood. (Mitchell., 1929b)
In the early stages of phys i o l o g i c a l i n v e s t i g a t i o n the
amino acids absorbed by way of the i n t e s t i n e were thought
to be almost immediately re-synthesized into proteins -
possibly i n the i n t e s t i n a l wall i t s e l f . Improvement and
perfection of methods for the detection of amino acids i n the
blood, however, l e d to the general discard of t h i s theory.
Rolin and Denis, (1912), were able to demonstrate f i r s t the
normal presence of amino acids i n the blood, and second, a
marked increase i n t h i s amino-acid content immediately a f t e r
protein ingestion by the animal. Further investigations
(Van Slyke, 1913) revealed the blood amino acid l e v e l to be
r e l a t i v e l y low even i n cases of animals fed diets r i c h i n
proteins or injected intravenously with amino acids, suggest
ing an almost immediate removal of these amino acids from
the blood by the ti s s u e s . Here again the procedure was
neither simple nor uniform: differences were observed i n
the rates of absorption of various i n d i v i d u a l amino acids
as well as differences i n retention by the various t i s s u e s .
(Van Slyke, 1942)
Functions of Nitrogenous Compounds i n the Tissues
Several possible fates may await the amino acids taken
up by the tissues. One immediately thinks of t h e i r re-synthe
s i s into body proteins to meet the requirements of growth i n
- 9 -
the young animal or of tissue repair and maintenance i n the
adult but these are not t h e i r only uses. A certa i n amount
of the amino acids, including those from protein consumed i n
excess of requirements, are deaminized and used as a source
of energy either immediately or at some l a t e r time. While
provision of an energy source i s not usually considered as
a major accomplishment of protein materials as contrasted
with carbohydrates and f a t s , i t seems l i k e l y that i t may
assume considerable proportions p a r t i c u l a r l y i n the case of
animals which subsist on high protein d i e t s .
The source of energy f o r muscular work,or conversely,
the influence of muscular work on protein metabolism has long
been a subject of controversy among phys i o l o g i s t s . At one
time i t was believed because of the nitrogenous nature of
the muscle tissues involved that protein i t s e l f supplied the
t o t a l energy required f o r t h i s metabolic phenomenon. This
view was discarded following completion of a nitrogen and
energy balance experiment i n Switzerland which i s of at l e a s t
s u f f i c i e n t h i s t o r i c a l i n t e r e s t to record. Two mountaineers,
•weighing s i x t y - s i x and seventy-six k i l o s respectively,
climbed the Faulhorn - a v e r t i c a l elevation of 1956 metres.
Their t o t a l protein consumption during the period of the climb
was found to be 22.09 gm. and 20.89 gm. which, even i f com
pl e t e l y u t i l i z e d could not supply the energy required f o r
performance of the work involved (Cathcart, 1925). Although
t h i s experiment could c e r t a i n l y not be judged s c i e n t i f i c a l l y
precise by modern standards, nevertheless i t s main conclusion
- 10 -
i s well founded that "The substances by the burning of which
force i s generated i n the muscles are not the albuminous
constituents of those tissues but non-nitrogenous substances
either f a t s or carbohydrates." More recent knowledge, as
indicated l a t e r , does not make such sweeping claims but
rather points to the use of protein under cert a i n conditions
f o r the supply of at leas t a portion of the body's energy.
I t may be of in t e r e s t to present at t h i s point a compari
son of the requirements of various animals i n order to i n
dicate the r e l a t i v e importance of protein as a dietary con
stit u e n t and as an energy source. Using the commonly accep
ted r a t i o of 2.00 mg. of endogenous nitrogen excreted per
Calorie of basal heat produced, (Ashworth, .Brody, Smuts,
Terroine and many others) the following protein requirements
might be expected to apply:
TABLE I: RELATIVE IMPORTANCE OF PROTEIN AS AN ENERGY SOURCE. Species Body Weight
(grams) B.M.R. (Cal/24hr)
Endogenous N Excretion mg.
(calculated)
Protein Equiv. gms.
Calories Supplied :
bv Protein
Species Body Weight (grams)
B.M.R. (Cal/24hr)
Endogenous N Excretion mg.
(calculated)
Protein Equiv. gms.
No. Rat 400 33.2 I 66.4 0.415 1.70 5.12
Cat 3000 152.0 304.0 1.90 7.80 5.13
Dog 14000 485.0 970.0 6.06 24.84 5.13
Sheep 45000 1160.0 2320.0 14.56 59.50 5.13
Man 65000 1640.0 3280.0 20^50 84.05 5.13
Basal metabolism data are taken from Benedict, " V i t a l Energetics" (1938). Calories supplied are calculated on the basis of Rubner's 4.1 Cal/gm.
- l i
l t i s i n t e r e s t i n g to note that i n a l l these animals the
amount of protein that must be fed i n order to s a t i s f y the
body's endogenous needs f o r nitrogen supply only approximately
f i v e percent of the t o t a l c a l o r i c requirements. The f a c t
that carnivora are included among these findings suggests
the p o s s i b i l i t y that such animals do not necessarily require
a large proportion of protein i n t h e i r d i e t and moreover
that they may have become meat eaters through reasons of
ecological advantage rather than phy s i o l o g i c a l necessity.
Such an observation appears e s p e c i a l l y pertinent with regard
to the formulation of p u r i f i e d rations f o r experimental use
with mink.
Further uses of the amino acids include the formation
of various enzymes, hormones and detoxication products such
as those formed by means of conjugation with glycine. I t i s
doubtful i n the course of any of these functions whether the
actual dietary amino acids are used as such. Rather, these
amino acids are modified through decarboxylation, deamination
and s i m i l a r processes and d i f f e r e n t fragments are re-conjugat
ed to give r i s e to the amino acids of the t i s s u e s . In the
course of these transformations some of the non-nitrogenous
residues may be converted to glucose, glycogen or f a t and
stored i n the animal body.
S p e c i f i c Dynamic Action
Any discussion of nitrogenous foods i n the l i g h t of
t h e i r energetic e f f i c i e n c y would be incomplete without men-
tion of t h e i r c h a r a c t e r i s t i c S p e c i f i c A A c t i o n . E a r l y
- 12 -
n u t r i t i o n i s t s , i n attempting energy balance t r i a l s , found
that when an amount of protein s u f f i c i e n t to meet an animal's
basal energy requirements was fed, i t raised the heat output
considerably over the previous l e v e l . Lusk (1931b) (15) i n
his laboratory has measured heat production of mature dogs
i n complete repose a f t e r consuming p u r i f i e d diets with the
following r e s u l t s :
100 c a l s . ingested as protein of meat increase heat production 30 c a l s .
100 c a l s . ingested as f a t increase heat production 4.1 c a l s .
100 c a l s . ingested as glucose increase heat production 4.9 c a l s .
These findings indicate that i n protein foods at l e a s t the
energy l o s s of SDA i s of s u f f i c i e n t magnitude to warrant
careful consideration i n c a l c u l a t i o n of n u t r i t i o n a l require
ments. The cause and nature of SDA has been the subject of
much intense i n v e s t i g a t i o n . Mere mechanical i r r i t a t i o n of
the i n t e s t i n a l t r a c t i s not the cause of l i b e r a t i o n of t h i s
waste energy as evidenced by the experiments of Benedict and
Emmes (1912) who fed humans cathartics and agar-agar with no
subsequent increase i n heat production. Further, the sugges
tion that SDA might be caused by the work of digestion was
discarded on the basis that amino acids injected into the
animal body raised the l e v e l of metabolism equally with a
s i m i l a r quantity of the same amino acids ingested by the
animal (Weiss, 1924). Another theory that amino acids act
as stimulants to c e l l u l a r metabolism has been rejected i n the
l i g h t of recent work by Borsook (1936). The most commonly
13 -
^accepted view today postulates that SDA arises as a r e s u l t
of intermediary chemical reactions undergone by the amino
acids and i s f a i r l y c l o s e l y correlated with the t o t a l energy
involved i n the metabolism of those amino acids (Kriss, 1941).
The SDA of proteins varies according to the consti.tu.egt amino
acids and the "balance" of those amino acids i n the l a r g e r
molecule. Maximum values for SDA are obtained at environ
mental temperatures of 25°C or over ; minimum values are
shown at low temperatures of about 0 - 5°C, i n d i c a t i n g that
the animal may make use of t h i s otherwise wasted energy to
maintain i t s body temperature i n cases of environmental ex
tremes. Apart from i t s purely t h e o r e t i c a l connotations, SDA
would appear to be of s i g n i f i c a n t importance i n the study of
mink n u t r i t i o n by reason of the facts that the normal mink
ra t i o n as now fed contains considerable protein and that the
environmental temperatures under which these animals are kept
are often below those minimums reported above.
Protein Storage
Actual storage of protein materials as such at f i r s t
considered highly improbable has more recently become a sub
ject f o r intense inves t i g a t i o n . P o s s i b i l i t y of protein
storage i n the animal body was postulated by Lusk (1931,c) i n
an attempt to explain the continued nitrogen excretion of
animals maintained 6n a nitrogen-free diet. The l a g i n
attaining nitrogen equilibrium a f t e r an increase or decrease
i n protein intake was observed by several investigators, i n
cluding Deuel (1928,a), Morrison (1942} and Ashworth and
- 14 -
Brody (1933) with the r e s u l t that protein retention has been
generally c l a s s i f i e d under two broad headings: (Koster l i t z ,
1946)
1. Nitrogen retained or l o s t i n conjunction with changes -
i n protein intake. This nitrogen takes on the form of l a b i l e
protein i n the cytoplasm of l i v e r and to some extent other
t i s s u e s .
2. A more stable type of protein storage evidenced i n
animals maintained on a protein-free d i e t or i n animals ex
posed to some abnormal nitrogen loss as i n cases of bleeding
or thermal burning.
More and more i n recent years the concept of the nature
of stored or deposit "protein has changed from the early p i c
ture of an i n e r t "store" to that of a dynamic equilibrium.
This l a t t e r view has been supported by the experiments of
Borsook (1943) and more recently of Schoe)ieimer (1942) who
made use of nitrogen isotopes as b i o l o g i c a l t r a c e r s . I t must
be emphasized here and i t v a i l become more evident l a t e r that
one of the more perplexing problems involved i n the study of
the nitrogen metabolism of any animal i s the correct evalua
t i o n of the protein reserves of the body.
While i t i s comparatively easy to theorize upon the
general scheme of protein metabolism, i t i s correspondingly
d i f f i c u l t to demonstrate the actual mechanics of the reactions
involved i n the various phases of the^operation. For instance,
one may point to protein synthesis i n the body as a mere con
jugation of amino acids, or indeed, as the reversible phase
- 15 -
of the reactions of p r o t e o l y s i s . In instances where the amino
acid content of the dietary constituents and the end products
of protein digestion are s i m i l a r , such may well be the case,
but i f , as i s often true i n animal n u t r i t i o n , "complete"
proteins are to be b u i l t from "incomplete", then obviously
a more detailed procedure i s involved. The work of Wastneys
and Borsook (19S5) has demonstrated the synthetic as well as
hydrolytic effects of the g a s t r o - i n t e s t i n a l proteases: pep
s i n and t r y p s i n . I t appears, however, that these two diverse
actions are regulated by r i g i d and s i m i l a r l y diverse condi
tions of temperature and pH, therefore, i t i s immediately
obvious that the optima of hydrolytic and synthetic action
cannot occur simultaneously. I t must be admitted that pro
t e i n synthesis i s an extremely complex procedure possibly
including successive reductions, oxidations and polymeriza
t i o n s .
Microbial Action
A supplementary phase i n the metabolism of proteins i s
carried out by the microorganisms l i v i n g within the alimentary
canal of the host animal. M i c r o b i a l digestion occurs to a
cer t a i n extent i n a l l animals i n the large i n t e s t i n e where
amino acids and other protein residues that have escaped
e a r l i e r absorption are exposed to b a c t e r i a l decarboxylation
to form t h e i r corresponding amines. Many of the amines so
formed are toxic, however, t h e i r creation i n a part of the
digestive t r a c t from which, at l e a s t i n the l i g h t of present
knowledge, absorption i s very s l i g h t , suggests that they have
- 16 -
but a doubtful b i o l o g i c a l significance ( C a h i l l , 1944,b).
The puocess of deamination may also be brought about by
microbial action; the method involved being apparently depen
dent upon conditions i n the i n t e s t i n e regarding oxygen supply,
a c i d i t y and the l i k e . I t i s i n t e r e s t i n g to record the f i n d
ings of Hanke and Koessler (1920) regarding b a c t e r i a l action
i n the intestine wherein they propose a buffering e f f e c t i n
herent i n such reactions. From t h i s work i t would appear
that production of amines from amino acids by bacteria occurs
only i n acid producing media while deamination r e s u l t s i n a
buffered or alkaline medium.
Another type of b a c t e r i a l a c t i v i t y that has recently
assumed prominence i n n u t r i t i o n a l studies i s that of protein
synthesis i n the digestive t r a c t s of some animals. In rumin
ants, f o r example, the rumen microflora are able to degrade
protein from the ingesta, l i b e r a t i n g ammonia. Other bacteria,
using t h i s ammonia as a s t a r t i n g point, are able to synthe
siz e proteins f o r t h e i r own use. The host organism, i n turn,
from the bodies of such bacteria, i s able to obtain s i g n i
f i c a n t amounts of protein additional to that produced through
the e f f o r t s of i t s own normal digestive a c t i v i t y . Possibly
the most s i g n i f i c a n t feature of t h i s b a c t e r i a l action occurs
i n cases where dietary protein i s low and hence where protein
synthesis must exceed degradation. In such instances, the
bacteria may u t i l i z e non-protein nitrogen sources as a basis
f o r t h e i r synthetic a c t i v i t y thus increasing the supply of
available nitrogen to the animal body (McNaught, 1947).
- 17 -
Apart from isuminants and those types of animals possessed
of naturally enlarged caeca, however, animals do not harbour
s u f f i c i e n t micro-organisms to produce s i g n i f i c a n t quantities
of amino acids and proteins. The r e l a t i v e l y simple type of
digestive t r a c t of carnivora greatly reduces the opportunity
for synthesis and increases the dependence of the animal upon
dietary sources of protein.
Endogenous Nitrogen Metabolism
The foregoing discussion has been concerned p r i m a r i l y
with the metabolism of nitrogenous materials entering the
body through the digestive t r a c t . Another aspect of t h i s
metabolism e x i s t s , namely the transfers involved among the
nitrogenous constituents of the tissues themselves. The
existence of t h i s mode of metabolism was made known through
the q u a l i t a t i v e differences i n the end products of protein
digestion found i n the urine of animals maintained at wide
l y d i f f e r e n t l e v e l s of protein intake.
Theories of Endogenous Nitrogen Metabolism
Numerous investigators pondered the significance of these
inconsistencies i n nitrogen excretion, however, i t was not
u n t i l the c l a s s i s work of F o l i n , (1905), that a workable ex
planation was reached. F o l i n noted two main types of nitrogen
excretion i n the urine - one constant, the other extremely
variable. The former types represented i n the urine by such
substances as creatinine and neutral sulphur, he termed en
dogenous nitrogen excretion. The l a t t e r , characterized by
- 18 -
formation of urea and inorganic sulphates, was c l a s s i f i e d as
exogenous nitrogen excretion. While the exogenous quota
appeared mainly concerned with the products of hydrolysis, the
endogenous portion was taken as representative of oxidations
occuring throughout the body tissues generally. F o l i n r s
theory with the exception of minor modifications occasioned
"by improvement i n techniques and subsequent advances i n the
knowledge of separation and structure of the compounds involv
ed has received wide acceptance by biochemists, at l e a s t u n t i l
very recently.
The concept of endogenous nitrogen as postulated by F o l i n
was broadened by l a t e r workers, including Lusk and Thomas, to
embrace variable quantities of a l a b i l e or deposit protein.
I t was noticed that animals fed nitrogen-free diets took a
certain length of time often several days to reach a base
(endogenous) l e v e l of nitrogen excretion i n t h e i r urine,
(Smuts, 1939), and that t h i s time was roughly proportional
to the nitrogen content of the pre-test d i e t . The thought
n a t u r a l l y arose that a temporary store of l a b i l e protein
existed i n the blood or c e l l u l a r f l u i d s of the body and was
drawn upon during periods of negative nitrogen balance.
S i g n i f i c a n t l y , the t r a n s i t i o n i n the products of nitrogen ex
cret i o n also lags behind the normal time of digestion when
protein i s once more included i n the d i e t , thus suggesting
the replenishment of t h i s nitrogen r e s e r v o i r .
E s s e n t i a l l y , the F o l i n theory of endogenous nitrogen ex
cretion presumes the existence of an e a s i l y mobilized yet
temporarily i n e r t store of nitrogenous material. More and
- 19 -
more i n recent years and e s p e c i a l l y following the investiga
tions of Borsook et a l , (1955) the concept of the so-called
"deposit protein" reservoir has changed to one of a dynamic
equilibrium. Borsook suggests the use of the term "continu
ing metabolism" with reference to the animal body's use of
protein reserves; drawing a sharj d i f f e r e n t i a t i o n between
t h i s quota and the "wear and tear" portion of F o l i n . Con
tinuing metabolism varies from one experimental animal to
another, depending upon the previous dietary h i s t o r y and i n
volves continuous processes of amino acid degradation and
synthesis.
Measurement of Endogenous Metabolism
This secondary nitrogen metabolism, i f i t may be termed
such i n contrast to that involving the dietary constituents
d i r e c t l y , i s obviously a reasonably constant measure of the
basal l e v e l of nitrogen excretion by any animal. Assessment
of basal nitrogen metabolism, (a convenient s t a r t i n g point
i n nitrogen balance studies, j u s t as basal metabolism i s i n
energetics) may be carried out by measurement of the c r e a t i
nine content of the urine of the experimental animals.
Creatinine, a waste product, i s without doubt the most t y p i
c a l l y endogenous produot of nitrogen metabolism as the asso
ciated neutral sulphur excretion i s not altogether independent
of dietary influences (Brody, 1954). Data have been produced
to show that creatinine output of i n d i v i d u a l animals kept
f i r s t on a high protein, l a t e r on an almost protein-free d i e t ,
i s p r a c t i c a l l y constant, (Hunter, 1928a), and although some
- 20 -
investigators such as Zwarenstein, (1926) claim to have noted
marked variations i n creatinine output; i t i s nevertheless
s i g n i f i c a n t that these variations cannot be correlated to
t o t a l nitrogen output. The exact role played by creatinine
i n metabolism i s yet to be described; however, Shaffer's
concept that creatinine i s a product and an index of one phase
of tissue catabolism (rather than of the e n t i r i t y ) and that
t h i s phase takes place l a r g e l y within the muscles, o f f e r s at
l e a s t a preliminary hypothesis (Shaffer, 1908).
Lest the urinary excretion of creatinine be taken as an
unimpeachable standard, several causes of v a r i a t i o n should be
l i s t e d . Hunter (1928b) notes that creatinine output i s pro
foundly influenced by continued absorption of unusual d i e t s .
For instance, i n humans fed a low protein, meat-free d i e t ,
the creatinine excretion declined gradually but s t e a d i l y . I t
seems possible that such a lowering of creatinine output might
be occasioned by a decline i n the t o t a l muscle mass. S i m i l a r
l y , animals maintained i n a f a s t i n g condition exhibit a slow
but rather regular f a l l i n creatinine excretion. Addition
of creatine, a precursor of creatinine to the d i e t may cause
Increased creatinine formation as may also the "pre-mortal"
r i s e a f t e r long periods of nitrogen starvation. In addition,
a v a r i e t y of pathological conditions may cause departures
from the normal l e v e l of creatinine output. Generally speak
ing, the constancy of endogenous catabolism should be evaluat
ed with respect to the exogenous catabolism rather than as an
absolute i n v a r i a b i l i t y .
- 21 -
As studies on the minimum endogenous nitrogen catabolism are commonly conducted upon animals fed protein-free or protein-low d i e t s , i t i s i n t e r e s t i n g to connect how these animals are able to maintain the i n t e g r i t y of t h e i r t i s s u e s . The mere fact that some nitrogenous materials are being excreted points to the continued d i s i n t e g r a t i o n of the body tissues yet obviously these tissues, i n certain s p e c i f i c cases at l e a s t , must be renewed from some source. In the early stages of protein i n a n i t i o n i t seems possible that the body's continued losses of nitrogenous materials may be borne by the blood supply but i n time t h i s supply must be renewed. Indeed, at a l l times the blood can be considered to have a "wear and tear" requirement i n the most l i t e r a l sense of the term. I t must be admitted that i n the l i g h t of present knowledge the ultimate o r i g i n of the endogenous portion of the blood's n i t r o gen remains a subject for speculation.
In summary of t h i s extremely short survey of the l i t e r a ture on. nitrogen and especially protein, metabolism, the features of exogenous and endogenous functions, of protein storage and of the constantly changing equilibrium e x i s t i n g among the nitrogenous constituents of the tissues must be emphasized. In an attempt to simplify to some extent an extremely complex picture and at the r i s k of appearing presumptuous, the wr i t e r has prepared the following schematic diagram of n i t r o gen metabolism w i t h i n the animal body.
INGESTION -PROTEINS, AMINO ACIDS, N.PN. i
-GASTRIC HYDROLYSIS-ENZYME ACTION PEPSIN, RENNIN... I
^INTESTINAL I BACTERIAL PUTREFACTION DECARBOXYLATION, DEAMIN-ATION BROUGHTABfaUT BY INTESTINAL MICROFLORA^
MICRO-BIOLOGICAL ACTION RUMINANTS-PROTEIN SYNTHESIS FROM AMMONIA, N.P.N. DIGESTION^
ENZYME ACTION TRYPSIN.EREPSIN FURTHER CLEAV-
v AGE BEGUN BY \ PEPSIN.
FAECAL EXCRETION UNDIGESTED PROTEIN & PROTEIN RESIDUES. EXCESS EXCRETIONS OF THE DIGESTIVE TRACT, AMMONIA.
INTESTINAL ABSORPTION LYMPH CIRCULATION-^ \
CATABOLISM DEAMIN ATION, DECARBOX-
UREA,
YLATJON
^-BLOOD CIRCULATION s~ CELLULAR ft PLASMA
S PROTEINS-^
>M ANABOLISNK ECARBOX- FORMATION OF-
mON ^ TISSUE PROTEINS. V . \ ^ENZYMES. HORMO^ESA CREATININES^: DETOXICAtlON PRODUCTS
CONVERSION OF EXCESS NON-NITROGENOUS RESIDUES, AND POSSIBLE USE FOR ENERGY SUPPLY. J
NITROGEN LOSS NITROGEN STORAGE
C Y T O P L A S M I C DEPOSITION OF PROTEIN IN LIVER, M U S C L E , & OTHER TISSUES
FIGURE 2 : NITROGENMETABOLISMiN T H E ANIMAL BODY
- 25 -
The Significance of Nitrogen Balance
, E a r l i e r i n t h i s paper reference was made to the form of
experimentation known as "nitrogen balance", that i s , the
comparison of the nitrogen content of the ingesta and excreta
of various animals, A condition of nitrogen equilibrium i s
said to exis t wherever the loss of nitrogen from an animal's
body equals the nitrogen content of i t s food during a s i m i l a r
measured i n t e r v a l of time. Where nitrogen excretion exceeds
intake, the animal under consideration i s termed i n negative
nitrogen balance, and conversely where nitrogen i s retained
i n the body ( i . e . where intake exceeds excretion), the animal
i s i n po s i t i v e nitrogen balance. Animals that are increasing
t h e i r muscular tissues generally do not excrete as much n i t r o
gen as they take i n . Such animals include the young (growing)
adults recovering from wasting diseases, animals undergoing
muscle building exercise, and pregnant females. Sherman (1941),
c i t e s experiments to show that the animal body tends to adjust
i t s p rotein metabolism to i t s protein supply, and that once
i t i s accustomed to any certa i n rate of protein metabolism,
an appreciable length of time i s necessary to e f f e c t a
material adjustment.
Calculation of the State of Nitrogen Balance
The state of nitrogen balance i s of course calculated by
a measurement of the nitrogen content of ingesta and excreta
of an animal over a defined period of time. Ingesta refers
to the food intake as under normal conditions the nitrogen
content of impurities i n the water used i s n e g l i g i b l e .
- 24 -
Excreta includes a l l those body wastes that might conceivably
contain nitrogen: the faeces, urine, sweat, skin brushings
and f a l l e n h a i r . Of these a l l but the f i r s t two names are
commonly considered i n s i g n i f i c a n t , however, they may assume
importance i n furred animals l i k e mink, e s p e c i a l l y during
the shedding season. Further reference w i l l be made to t h i s
p o s s i b i l i t y l a t e r .
Many experiments and e s p e c i a l l y those dealing with n i t r o
gen minima are concerned s o l e l y with the urinary portion of
the excreta and t h e i r r e s u l t s are tabulated as such. The
general scheme of nitrogen balance t r i a l s involves f i r s t the
attainment of a minimum l e v e l of nitrogen excretion and second
the creation of nitrogen equilibrium through administration
of nutrients of known nitrogen content.
Two major requirements must be met i n the attainment of
minimum nitrogen excretion:
1. Protein intake should be lowered, preferably to zero, -
or at l e a s t to such a l e v e l as w i l l not influence the rate of
nitrogen excretion by the kidneys. Such a l e v e l w i l l , of
course, vary with the nature of the dietary protein.
2. Adequate energy intake should be provided through
non-protein sources i n order to prevent the catabolism of
nitrogenous constituents of the tissues as f a r as possible.
In order to ensure an adequate c a l o r i c intake i t has been
shown expedient to feed carbohydrates and fa t s i n amounts
considerably greater than the actual basal metabolism would
indicate necessary.
The value of nitrogen balance experiments l i e s i n t h e i r
- 25 -
applicati o n i n the study of the protein requirements of
animals. I t might be supposed that to meet an animal's basal
needs f o r nitrogenous material a l l that would be_nejDjeaaary^"
would be an amount equal to that l o s t by the animal under
the conditions of minimal excretion previously described.
Such i s not the case, however, due mainly to the diverse
composition of the various native proteins that may be fed.
Accordingly, modern nitrogen balance work has favoured the
use of protein hydrolysates or amino acid mixtures as nitrogen
sources on the assumption that the mixture supplying propor
tions of amino acids nearest the animal's requirements w i l l
maintain nitrogen equilibrium at the lowest possible l e v e l
(Kade, 1948), Such experiments serve a twofold purpose i n
that they provide valuable data regarding amino acid composi
t i o n of various feed combinations i n c i d e n t a l to the informa
t i o n obtained on the state of nitrogen metabolism i n the
animal.
N u t r i t i v e Value of Proteins
In order to properly appreciate the findings of nitrogen
balance experiments, consideration must be made of the net
value of d i f f e r e n t proteins to the animal under consideration.
This concept of net worth of proteins has been commonly ap
proached under the heading of B i o l o g i c a l Value - an extremely
important phase of p r a c t i c a l n u t r i t i o n . While i t i s r e a d i l y
acknowledged that experiments concerned with i n d i v i d u a l amino
acid relationships are indispensable from the point of view
of fundamental knowledge, nevertheless such findings must be
- 26 -
supplemented by an accurate appraisal of the a v a i l a b i l i t y of
such amino acids to the animal to be of much p r a c t i c a l use.
Higher animals have depended i n the past upon native proteins
as the main source of t h e i r dietary nitrogen, and i n a l l pro
b a b i l i t y w i l l continue to do so i n the future. Consequently,
an experimental balance must be struck: chemical and b i o l o
g i c a l or a comparison of the precise requirements of an animal
with the combinations that are l i k e l y to exi s t i n i t s natural
dietary sources.
The absolute e f f i c i e n c y of proteins i n feeds, that i s to
say t h e i r b i o l o g i c a l values, may be expressed as the percen
tage of t o t a l intake of these nutrients a c t u a l l y u t i l i z e d by
the body. A workable equation f o r the expression of such
values i s that originated by Thomas i n 1909, l a t e r modified
by M i t c h e l l (1924) as follows:
N intake - (faecal N - metabolic N) - ( u r i n a r y N - endogenous N)xl00 N intake - (faecal N - metabolic N)
This formula i n general use takes cognizance of the f a c t
that the endogenous or metabolic portions of the t o t a l n i t r o
gen available have been u t i l i z e d by the body even though
they are subsequently excreted.
As examples of b i o l o g i c a l values, the following table i s
quoted from Maynard (4£): TABLE l i t BIOLOGICAL VALUE OF THE PROTEINS OF HUMAN FOODS Food B i o l . Value <fo Food B i o l . Value i Whole egg 94 Whole wheat 67
85 Potato 67 Egg white 83 Rolled oats 65 Beef l i v e r 77 Whole corn 60 Beef heart 74 Wheat f l o u r 52 Beef round 69 N a v ^ lasted] 38
- 27 -
Several features contribute to the ultimate b i o l o g i c a l
value that w i l l be assigned d i f f e r e n t proteins. B r i g f l y ,
these include the relationship e x i s t i n g among the constituent
amino acids, ( e s p e c i a l l y as regards the "essential"amino
acids) the proportion of the protein moeity to the remainder
of the d i e t and the s t r u c t u r a l composition of the entire food
as related to ease of digestion. These factors seem of s u f f i
cient importance i n the determination of the n u t r i t i v e value
of proteins to j u s t i f y the i n c l u s i o n of a b r i e f discussion.
In the past, amino acids have been broadly c l a s s i f i e d
under two main headings variously known as " e s s e n t i a l " and
"non-essential" or "indispensable" and "dispensable". The
essential type include those amino acids that cannot be syn
thesized i n the animal body i n s u f f i c i e n t quantity to meet
the requirements for them and hence must form an indispensable"
portion of the di e t . The non-essential amino acids are of
course those which can be manufactured from other sources
within the animal body. This l i n e of thought proposes that
the b i o l o g i c a l value of any absorbed protein i s dependent on
the proportions of esse n t i a l amino acids which i t contains.
For example, i f one e s s e n t i a l amino acid i s completely lacking
from a protein, i t w i l l prevent the f u l l u t i l i z a t i o n of the
other amino acids and thus w i l l s e r i o u s l y lower the net value
of that protein to the animal. I t i s in t e r e s t i n g to record
that i n recent years the i n d i s p e n s a b i l i t y of at l e a s t some
amino acids has been attributed to not the nitrogenous por
t i o n but rather the configuration of the elements carbon,
hydrogen, and oxygen. Rose (1937) has demonstrated that
- 28 -
phenyl pyruvic acid may take the place of pSylalanine i n which
case an animal could probably convert some of i t s pyruvic acid
to the corresponding amino a c i d . E v e n so, one might be excus
ed i n naming pSylalanine e s s e n t i a l on the -grounds that the
u t i l i z a b l e pyruvic acid i s not i t s e l f a natural component of
foods.
Among the naturally-occurring proteins, those composing
the endosperm of cereal grains are considerably lower i n some
of the esse n t i a l amino acids, notably l y s i n e , than most pro
teins of animal o r i g i n . Due to t h i s inconsistency, an erro
neous b e l i e f has been founded that plant proteins i n general
are unbalanced and hence i n f e r i o r . T h i s concept i s untrue as
many proteins from the l e a f y parts or the embryos of plants
are b i o l o g i c a l l y equal and economically superior to animal
proteins: a f a c t that i s important i n the formulation of
rations both experimental and p r a c t i c a l . I n b r i e f , the com
binations that may e x i s t among the constituent amino acids
i n any native protein are so diverse that generalization as
to t h e i r n u t r i t i v e q u a l i t i e s i s unsafe.
The e f f i c i e n c y of proteins i n meeting the requirements
of i n d i v i d u a l animals i s also dependent i n no small measure
upon the accompanying non-nitrogenous portions of the r a t i o n .
B o t h carbohydrates and fats are able to diminish the cata
bolism of proteins, that i s , exert a "protein-sparing" action
carbohydrates apparently being more e f f i c i e n t than f a t s i n
th i s regard. The actual mechanics of t h i s action are not
ea s i l y described. I t would appear that i n eases where animals
do not receive s u f f i c i e n t carbohydrate to maintain the normal
- 29 -
glucose content of t h e i r blood the required glucose may be
supplied by deaminized residues of amino acids (Landergren,
1907). A supply of carbohydrate i n such instances would of
course avoid the use of proteins i n the formation of blood
sugar and might be ef f e c t i v e at any l e v e l of intake due to
the r e l a t i v e ease of oxidation of glucose. In a s i m i l a r man
ner f a t may spare protein by preventing i t s consumption f o r
energy purposes. The lower e f f i c i e n c y of f a t may be explain
ed by the f a c t that f a t stores i n the body are less r e a d i l y
depleted than are those of glycogen ( H i l l , 1924).
The preceding discussion has stressed the importance of
the chemical properties of the components of a r a t i o n i n the
determination of i t s n u t r i t i v e value. Attention must also
be paid to the physical properties of the dietary mixture, as
unless the nutrients can be made available f o r absorption,
they oannot be u t i l i z e d i n any way by the animal. A simple
example of such a condition may be found i n the ooarse dry
roughage feeds which made up a considerable proportion of the
ration of herbivores. Ruminants and kindred types of animals
are able, by reason of fermentation processes c a r r i e d out i n
enlargements of t h e i r digestive t r a c t s , to s p l i t away the
tough, c e l l u l o s e - r i c h sheath which protects the natural proteins
of suoh forage. Those animals possessed of a simple digestive
t r a c t , (and esp e c i a l l y carnivores) are unable to e f f e c t t h i s
preliminary digestion, and are thus faced with the uncomfor
table s i t u a t i o n of having a supply of chemically-suitable
proteins that they are unable to assimilate. Taking another
extreme example, keratin, the protein of h a i r i s strongly
- 30 -
r e s i s t a n t to digestion and therefore must he considered of low
b i o l o g i c a l value. I t can furnish but a n e g l i g i b l e amount of
use f u l nitrogenous material to the body again f o r reasons of
physical rather than chemioal structure. I t w i l l be evident
from the b r i e f discussion of these two examples that two main
factors play a part i n the physical aspect of the values of
proteins: the actual gross structure of the food material
and the species,differences which determine the digestive
c a p a b i l i t i e s of the various animals.
The foregoing pages should be considered i n the nature
of a preois, almost an abstract, of the extremely extensive
and involved l i t e r a t u r e dealing with the metabolism of n i t r o
genous compounds. Many other features might have been consi
dered, including the varying requirements f o r proteins during
the successive stages of growth and development and the phy
s i o l o g i c a l c h a r a c t e r i s t i c s , both normal and abnormal. The
i n c l u s i o n of such material, though i t i s c e r t a i n l y relevant,
would not add m a t e r i a l l y to the underlying theme of t h i s
thesis, namely the in v e s t i g a t i o n of the basal nitrogen meta
bolism i n adult mink.
- 31 -
EXPERIMENTAL
A perfect experiment i n any f i e l d of science may be said to be one that has been planned and conducted i n such a way that the results obtained are susceptible of only one i n t e r p r e t a t i o n .
- H. H. M i t c h e l l
Some Considerations Involved i n Planning a N u t r i t i o n Experiment
Several d i f f e r e n t methods have been attempted i n studies
of nitrogen metabolism with varying degrees of sucoess i n
operation. I t i s desirable before planning an experiment i n
volving t h i s subject to weigh the advantages and disadvantages
of such methods as discussed i n the l i t e r a t u r e and evaluate
them i n the l i g h t of c e r t a i n general considerations which must
be met to ensure successful r e s u l t s .
A preliminary e s s e n t i a l of any n u t r i t i o n a l experiment -
indeed, any experiment - i s that of provision of adequate
" oontrols." To obtain r e s u l t s that may be applied to normal
animals, i t i s evident that one must work with normal animals
either as the actual experimental subjects or as controls f o r
comparison. This i d e a l of normality, so l o g i c a l i n theory ,
i s extremely d i f f i c u l t to a t t a i n i n practice and f a i l u r e of
i t s attainment i s perhaps the outstanding contributory cause
to experimental f a i l u r e .
Many instances may be c i t e d of the dangerous breaches i n
experimental data occasioned by i n s u f f i c i e n t attention to
the aspect of normality. One of the most pertinent discus
sions of t h i s topic i s presented by Baldwin (1947,b) as follows:
- 33 -
In an i n t a c t , normal animal, to take a s p e c i f i c example, we cannot obtain much information about the metabolism of proteins by straightforward i n vestigation of nitrogenous substances entering and leaving the organism. I f proteins are fed to a mammal, we f i n d that the ingoing protein nitrogen emerges again i n the form of urea or i n a b i r d i n that of u r i c a c i d . Very l i t t l e more can be d i s covered. How the nitrogen i s detached from the protein and how i t i s b u i l t up into urea i n the one case into u r i c acid i n the other, we cannot discover without taking the animal more or le s s to pieces. I f , however, we take a mammal from which the l i v e r has been removed, i t w i l l survive f o r some days provided that proteins are witheld from the d i e t . I f a protein meal i s given, however, the animal quickly d i e s . Closer examinat i o n reveals that death i s due i n the main to poisoning by ammonia and that the blood and urine a l i k e contain ammonia but no urea. But whereas ammonia set free by deamination i s converted into urea i n the normal animal, urea production ceases with hepatectomy.
This i s o l a t e d example i l l u s t r a t e s a ptly the need f o r some
sort of derangement or abnormality on the one hand i n the i n
vestigation of fundamentals of metabolism,coupled with the
necessity f o r careful i n t e r p r e t a t i o n i n the p r a c t i c a l applica
t i o n to normal animals on the other. In countless cases erro
neous conclusions have arisen as a r e s u l t of abnormalities
introduced i n the experimental animals or as a r e s u l t of the
experimental techniques adopted. Indeed, many of these ab
normalities were necessary i n the conduct of the investigations;
the point of the matter i s that they ,were not recognized as
such and so considered i n the f i n a l compilation of the data.
A delicate balance i n experimental approach appears
necessary between i n v i t r o and i n vivo techniques. Any of the
questions posed by phenomena of intermediary metabolism can
not be adequately answered at the present time at l e a s t by
experiments upon i n t a c t animals. By the same token, however,
- 33 -
i t must not be accepted that simply because a reaction takes
plaee i n a P e t r i dish or t e s t tube, i t w i l l produce the same
re s u l t s i n a l i v i n g organism. A combination of data i s i n
dicated, taking into consideration those gathered i n varying
types of experiments, by d i f f e r e n t workers under diverse
laboratory conditions; and above a l l i n c l u s i v e of s u f f i c i e n t
numbers to be s t a t i s t i c a l l y s i g n i f i c a n t .
Choice of F i e l d f o r Experimentation
In planning an experiment on the n u t r i t i o n a l requirements
of mink, one must always bear i n mind the almost complete
lack of a background of s c i e n t i f i c research with these animals
as contrasted to other species. From a p r a c t i c a l standpoint
too the widespread v a r i a t i o n and disagreement exi s t i n g i n the
composition of rations on economically successful mink ranches
lends weight to the aura of uncertainty surrounding the physio
l o g i c a l needs of these animals. I t seems evident, therefore,
that any preliminary inves t i g a t i o n should be directed toward
attainment of the correct balance both economic and physio
l o g i c among those substances which are to form the major por
t i o n of the d i e t , namely the carbohydrates, fa t s and proteins.
In t h i s s p e c i f i c instance of experimentation the inves
t i g a t i o n of protein requirements was undertaken because t h e i r
Importance qua n t i t a t i v e l y as a r a t i o n constituent seems en
hanced by the p e c u l i a r p h y s i o l o g i c a l c h a r a c t e r i s t i c s of the
animal. Further, while the modern highly successful plan of
investigating i n d i v i d u a l amino acid requirements might have
been indulged i n , a note of caution seemed wise. Accordingly,
- 54 -
a plan of experimentation was drawn up dealing with the gross
or o v e r a l l picture of protein usage as indicated by means of
various nitrogen balance t r i a l s .
Plan of Experiment
The plan of experiment adopted may be divided into essen
t i a l l y two parts. F i r s t , a t r i a l was designed using two
animals i n which the absolute minimum of urinary nitrogen ex
cretion was measured i n adult animals maintained i n a f a s t i n g
condition. This preliminary i n v e s t i g a t i o n was believed nec
essary f o r the determination of the basal or minimal l e v e l of
nitrogen excretion and as a guide to the length of time nec
essary to reduoe the body's main store of e a s i l y mobilized
or l a b i l e protein. In addition, i t was f e l t that a compari
son of data obtained i n this way with the minimal figures f o r
other species available i n the l i t e r a t u r e might be of great
assistance i n an estimation of the basal metabolism of mink.
The second and more extensive part of the experiment i n
volved tabulation of nitrogen balance data using as many
animals as possible with the apparatus and time a v a i l a b l e .
The aim of thi s d i v i s i o n of the experiment was to maintain
a status of nitrogen equilibrium on a die t containing the
minimum possible nitrogen content. I t was important to produce
nitrogen equilibrium at t h i s basal l e v e l because as suggest
ed i n the e a r l i e r review, protein i s not stored to any appre
ciable extent i n the adult animal and hence i t i s altogether
possible that equilibrium might be established at a higher
than normal l e v e l . The information gathered i n t h i s way would
- 35 -
i t was hoped, serve as an accurate index of the protein re
quirements of the animal, at l e a s t insofar as the s p e c i f i c
protein used i n the experimental r a t i o n was concerned. Further,
i t was hoped that i n combination with basal metabolism data,
the information r e s u l t i n g from t h i s experiment might serve as
an i n d i c a t i o n of the t o t a l c a l o r i c needs of the animal. Crea
ti n i n e nitrogen determinations as well as t o t a l nitrogen de
terminations were made during t h i s l a t t e r period of experi
mentation as a form of check on the b i o l o g i c a l value of the
protein used (Murlin, 1948),
Method:
The search f o r an absolute minimum i s l i k e the philosophers 1 search f o r the absolute truth.
- E. P. Cathcart
Actually, there i s not one minimum but several protein
minima concerned with many factors that must be considered
c a r e f u l l y when la y i n g down the method of experimentation
(Melnick, 1936). These factors include:
a. The nature of foodstuffs fed with the protein. b. The completeness of the d i e t - q u a n t i t a t i v e l y
and q u a l i t a t i v e l y . c. The c a l o r i c value of the food. d. The stage of maturity of experimental animals. e. The a c t i v i t y of the experimental animals. f . The environmental temperature. g. The n u t r i t i v e condition of the animals p r i o r
to the t e s t combined with adequate preliminary adjustment.
These items give some hint of the precautions necessary
i n setting up an experiment of t h i s type and at the same time
serve as a warning against a too hurried i n t e r p r e t a t i o n of
r e s u l t s
- 36 -
C e r t a i n f e a t u r e s o f t h e method a d o p t e d were common t o
b o t h b r a n c h e s o f t h e e x p e r i m e n t and t h e s e w i l l be d i s c u s s e d
f i r s t . I t must be emphas ized f r o m the o u t s e t t h a t f o r l a c k
o f any p r e v i o u s d a t a on the s u b j e c t , c o n s i d e r a b l e e x p e r i m e n t a
t i o n was n e c e s s a r y i n t o the c o n s t r u c t i o n o f w o r k a b l e a p p a r a t u s .
The e x p e r i m e n t a l a n i m a l s were m a i n t a i n e d i n a s e p a r a t e room
f rom t h e m a i n c o l o n y o f t h e U n i v e r s i t y F u r A n i m a l U n i t and
were t h e r e f o r e c o m p l e t e l y q u i e t and u n d i s t u r b e d e x c e p t f o r
t he s h o r t p e r i o d d a i l y when u r i n e c o l l e c t i o n s were made and
f e e d i n g , i f a n y , c a r r i e d o u t . E x t r e m e s o f t e m p e r a t u r e were
g u a r d e d a g a i n s t b y adequate v e n t i l a t i o n and a v o i d a n c e o f any
d i r e c t d r a u g h t s o f a i r a c r o s s t h e a n i m a l c a g e s . The cages
t h e m s e l v e s were o f s u f f i c i e n t s i z e t o a l l o w f r e e movement t o
t h e a n i m a l s y e t were c o n s i d e r a b l y s m a l l e r t h a n t h e n o r m a l
r a n c h u n i t i n o r d e r t o r e s t r i c t t h e i r a c t i v i t y t o n e a r e r b a s a l
c o n d i t i o n s .
U r i n e c o l l e c t i o n s were made by means o f s t a i n l e s s s t e e l
f u n n e l s o v e r w h i c h the cages were suspended e q u i p p e d w i t h w i r e
mesh f a e c e s s c r e e n s and g l a s s w o o l f i l t e r s . G r a d u a t e d c y l i n d e r s
(100 m l . ) were u s e d as c o n t a i n e r s f o r t h e u r i n e , a l l o w i n g f o r
r a p i d and r e a s o n a b l y a c c u r a t e d e t e r m i n a t i o n o f t h e t o t a l volume
o f e x c r e t i o n . U r i n e c o l l e c t i o n s were made d a i l y , u s i n g a t h i n
o v e r l a y o f t o l u e n e i n the g r a d u a t e s as a p r e s e r v a t i v e . I n
c a se s where samples were h e l d o v e r f o r a n a l y s i s , t h e y were
m a i n t a i n e d u n d e r r e f r i g e r a t i o n , a g a i n u s i n g t h e l a y e r o f t o l
uene t o e x c l u d e a i r . T h r o u g h o u t a l l e x p e r i m e n t s an ample
s u p p l y o f d r i n k i n g w a t e r was k e p t c o n s t a n t l y b e f o r e the a n i m a l s .
D u r i n g t h e n i t r o g e n b a l a n c e t r i a l , c o n s i d e r a b l e d i f f i c u l t y
- 37 -
was encountered i n the preparation and administration of syn
t h e t i c rations. Again, the lack of previous information on
the subject hampered investigations and several successive
mixtures were attempted before a successful r a t i o n was found.
In the preparation of the r a t i o n attention had to be paid not
only to the chemical composition, p a r t i c u l a r l y as regards
nitrogen content, but also to the physical nature as the con
sistency of the feed appeared of considerable importance i n
assuring the desired l e v e l of intake. Administration too pre
sented i t s problems as a method had to be devised whereby the
amount consumed by each animal could be accurately measured.
Two workable schemes were devised i n t h i s connection. In the
f i r s t , a semi-solid mixture was made of the ra t i o n , using d i s
t i l l e d water as a diluent and the r e s u l t i n g paste was extrud
ed to the animals through a hard glass tube. This method was
found s a t i s f a c t o r y with rations wherein starch was the dominant
carbohydrate as i t formed an adhesive g e l - l i k e mixture; how
ever, rations high i n sucrose were apt to go at le a s t i n part
into solution and another method had to be devised i n order
to avoid losses. The second type of feeder consisted of an
attachable container with a projecting l i p to prevent inac
curacies i n estimation of feed intake caused by the itfink's
natural habit of carrying o f f i t s feed before consuming i t .
Details of the experimental r a t i o n composition and methods of
administration are given at length i n Appendix I I .
Analysis of urine samples f o r t o t a l nitrogen was made
d a i l y i n the Animal N u t r i t i o n Laboratory using the Gunning
Modification of the Kjeldahl method. I t was noticed early i n
- 38
the course of experimentation that considerable quantities of
h a i r and skin debris were shed by the animals. The p o s s i b i l i t y
of an increase i n urinary N due to washing over t h i s matter
suggested i t s e l f . An experiment was designed, therefore, to
check nitrogen analyses of s i m i l a r samples before and a f t e r
contact with such extraneous matter. Check tests were made
from time to time f o r the detection of b i l e i n the urine as
th i s would of course indicate a difference- i n the physiologic
nature of the nitrogen content and f o r a s i m i l a r reason tests
for albumin were carr i e d out p e r i o d i c a l l y .
Creatinine determinations were made spectro-photometri-
c a l l y , using a modified a l k a l i n e p i c r a t e procedure o r i g i n a l l y
suggested by F o l i n and Jaff e (Peters, 1942). As previously
mentioned, these tests were not run d a i l y but were made at
def i n i t e i n t e r v a l s during the course of the experiment. As
reference has been made i n the l i t e r a t u r e that glucose may i n
terfere with the Jaff e reaction, (Barclay, 1947), Benedict's
tests were performed from time to time as a check on the v a l i
d i t y of the creatinine figures. The actual laboratory proce
dures adopted, including any modifications employed, are l i s t
ed i n Appendix I.
- 39 -
Observations and Discussions:
Endogenous Nitrogen Excretion
The data gathered i n the preliminary experiment (that i n
volving the f a s t i n g catabolism) are presented i n summary form
i n Table I I I . A "nitrogen c o e f f i c i e n t " , arrived upon by d i v i d
ing the t o t a l d a i l y urinary nitrogen excretion ( i n grams) by
the body weight ( i n kilograms) i s employed to give a more com
parative picture of the wastage of the protein reserves of the
body. The body weight f o r purposes of these calculations was
taken as the mean between s t a r t i n g and f i n i s h i n g weights.
A graphioal representation of the urinary nitrogen excre
t i o n of the two animals involved i n t h i s experiment i s present
ed i n figure 3 (a). The f i r s t day's urinary nitrogen loss by
animal number 1 i s indicated by a broken l i n e on the graph, as
i t was believed to be abnormally high due to faecal washing.
This possible source of error was immediately corrected by i n
s t a l l a t i o n of a succession of f i l t e r s as previously described.
I t w i l l be noticed that a preliminary f a s t of 9 days was
carried out i n order to overcome the effects of any previous
feeding. Although t h i s period may seem lengthy i n comparison
to those adopted f o r other species by Smuts (1935) i t was f e l t
j u s t i f i e d due to the absence of any p r i o r information i n t h i s
regard f o r the mink. After t h i s preliminary f a s t , a nitrogen-
free d i e t was administered i n an attempt to s a t i s f y the animals'
c a l o r i c requirements from some non-protein source. As a point
of i n t e r e s t i t may be recorded that, while animal number 2
died a f t e r 12 days on experiment, animal 1 continued f o r 20
days, a f t e r which i t was removed and returned to the Fur
- 40 -
Animal Unit i n apparent good health.
TABLE I I I : ENDOGENOUS NITROGEN EXCRETION DATA FOR MINK ON A NITROGEN-FREE DIET.
Mink No. 1 Mink No. 2 Day Total T o t a l N Coeff. Total Total N Coeff. Day
Urine N gms. Urine N gms. ml. gms. ml. gms.
1 105 B745 4.62 35 1.28 1.41 2 45 1.78 1.52 38 0.98 1.75 3 17 0.40 0.34 29 1.15 1.21 4 15 0.27 0.23 86 0.65 0.71 5 19 0.33 0.28 68 0.86 0.95 6 25 0.24 0.20 88 0.65 0.71 7 16 0.28 0.24 96 0.82 0.90 8 24 0.24 0.20 88 0.77 0.85 9 31 0.43 0.37 95 0.52 0.57
10 64 0.68 0.58 101 0.72 0.79 11 36 0.36 0.31 110 0.60 0.66 12 48 0.52 0.44 110 0.60 0.66 13 50 0.49 0.42 14 51 0.52 0.44 15 121 0.42 0.36 16 56 0.49 0.42 17 49 0.53 0.45 18 47 0.50 0.43 19 47 0.65 0.55 20 74 0.54 0.46
I t i s i n t e r e s t i n g to note that, i n common with other
species, the urinary nitrogen excretion f o r mink dropped o f f
sharply during the early stages of f a s t i n g and then tended to
l e v e l o f f on a more-or-less basal l e v e l . A d e f i n i t e increase
i n t o t a l nitrogen excretion was noticeable subsequent to the
administration of a nitrogen-free d i e t due probably to the
animals' increased water consumption and increased urinary ex
cretion occasioned by resumed ingestion of food. This increas
ed urine production by animals receiving feed a f t e r a f a s t was
most noticeable both i n t h i s and the l a t t e r section of t h i s
experiment and apparently outweighs any sparing e f f e c t that
the carbohydrates and fats fed might have had upon the dimin
ishing protein reserves of the body. I t i s perhaps s i g n i f i c a n t
- 4 1 -
to record that of the many nitrogen excretion studies examined
i n the l i t e r a t u r e , including those widely quoted works of
Brody and Smuts, none l i s t e d f i g ures on t o t a l urine volume.
One cannot help but wonder under the circumstances whether
many of the variations noted i n urinary nitrogen excretion
might not be due to fluctuations i n t h i s basic physical f a c t o r .
Tor purposes of comparison, nitrogen excretion figures c i t e d by
Lusk (52) from a starvation experiment and Deuel (53) from a
nitrogen-free d i e t t e s t are presented i n fi g u r e 3 (b). Here
the r e l a t i v e l y longer i n i t i a l period of sharply d e c l i n i n g n i t r o
gen excretion i s probably accounted f o r by the greater body
size of the experimental subjects.
During the mink experiment, a s l i g h t r i s e i n t o t a l urinary
nitrogen excretion may be noticed throughout the basal period.
This increase i s extremely gradual and gives no suggestion of
the so-called "pre-mortal r i s e " even i n the case of animal
number 2 which did die on experiment. An explanation of t h i s
increased nitrogen l o s s i s d i f f i c u l t , however, i t seems possible
that i t may be i n the nature of a compensatory reaction brought
about to meet the acidosis caused by the catabolism (and i n
complete oxidation) of body f a t .
As time permitted the use of two animals only on the pre
liminary nitrogen depletion phase of experiment, the r e s u l t s
cannot be regarded as conclusive by any means; however, they
do indicate c e r t a i n trends which may be of some s i g n i f i c a n c e .
Considerable v a r i a t i o n existed i n the nitrogen c o e f f i c i e n t s
exhibited by the two animals although the general excretion
eurves were reasonably s i m i l a r , (see f i g . 3A). I t appears
- 42 -
that a reasonably stable l e v e l of nitrogen excretion i s reached
a f t e r 4 days of f a s t i n g , and t h i s finding i s i n agreement with
the calculations published f o r other animals from M i t c h e l l ' s
laboratory. The establishment of an absolute basal l e v e l of
nitrogen excretion i s more d i f f i c u l t to a t t a i n and while that
plotted f o r animal number 1 (figure 3A) was reasonably constant,
ce r t a i n discrepancies do e x i s t . The data appear to suggest,
i n accordance with the theory of Borsook, that there i s no
cle a r cut "endogenous" l e v e l of nitrogen excretion but rather
a constantly changing nitrogen equilibrium which i s adjusted
to the nitrogen metabolism of the animal.
The maintenance of the l a r g e r animal i n a state of protein
i n a n i t i o n was accomplished with X4D3 apparent hardship on the
animal i t s e l f . The smaller animal, on the other hand, appear
ed to lose i t s v i t a l i t y rather quickly and as noted died during
the course of the experiment. This difference seems l i k e l y due
to the greater reserves of adipose tiss u e i n the former animal.
While both mink refused a considerable proportion of t h e i r
nitrogen-free energy source during the i n i t i a l stages of feed
ing, the la r g e r animal was able to meet at l e a s t part of i t s
c a l o r i c requirements through the breakdown of body f a t .
Nitrogen Balance Experiments
To r e i t e r a t e b r i e f l y , the scheme of the nitrogen balance
t r i a l s was to maintain nitrogen equilibrium at the lowest
possible l e v e l , using d i f f e r e n t s p e c i f i c nitrogen sources.
The animals were f i r s t fasted u n t i l c a l c u l a t i o n of t h e i r n i t r o
gen excretion indicated that they had reached a reasonably con
stant l e v e l ; they were then fed a nitrogen-free basal r a t i o n
TABLE IV (A) URINARY NITROGEN EXCRETION
ANIMAL NO: I
s
o g
S3 S3 S3 3 i l l I ^ 5 j-
I R 5 H
I B
IESI I E 9
raBlTOIBIIWllSElKS IB9SB
I KB ESI
i Egg
• B 9
IE9I
844 815 190
56?, 390 T£2 4001 3991 740
7 5 5
77S 790
310 303 | 755 283 2951 715 1781 133 U75 183 SloU'52
80 95 565 190 USOI56D S<32 482 545 35B S19 510 339 438 485 317 418 440
319 4301455
327 4$t> 366 4oj 814 875 358 735 398 « o 710 401 507|68? £50 324 645 118 157 U o 8 200 %oZ OX
Goo 115 148 595
2 6 4 5^0
585 226 283 580 540 £66 573 816 local 570 1140 1386
1KB IH 789 •• " 743 « 7511 «
7£o
E2B3I
cC
o
760 547 756 552 735 530
lg»iu!S]
781 B 740 « 700
623 » too BLI^ lo^ 570 « 103 547 « Uo5 2̂0 - 2,05
49o| » ho?
S3 EH 1551
103 105 103 ICS
BL £0S
191 W l 312,
440 415
618 560 540 >• 530 « 505 BLrt 103 490 ID'S 485 « 103 477 '« ICS 470 <» 10? 460 "BL 205 457 » i f t i 450 - 1<J1 442 .. y\\ 437 •« S?& 430 »
SB 59 SSBS
1160 1148
11%
1113 Vt&S 1V68
1130
1130 loto
352 255 466 35C 4 % mo 487 276 228 222 269 29€ 24ft
546 Z<J8 276IZ40 317 26o
258 273 251 2AO 213 167 148 159 141 163
205 157 148
171 174 15o1152
am
20f 17-a" 176 2-SI 2-3?,
399 10O0 'i
S83 383 5 3 2
584 584 855
3£f<93c 5"6o
.VEX TO F^£^UHC>-
T 1 I4*r| looo
- 43
and again fasted to confirm the r e s u l t s obtained previously.
Following t h i s i n i t i a l preparation, the mink were fed an amount
of the. nitrogen-free r a t i o n s u f f i c i e n t to meet t h e i r c a l o r i c
requirements as estimated from t h e i r body weights together
with a measured amount of protein food i n an attempt to reaoh
and maintain a state of nitrogen equilibrium. The oomplete
picture of urinary nitrogen excretion obtained as a r e s u l t of
these t r i a l s i s presented i n table IV" A. I t w i l l be noticed
that i n many oases a time l a g existed i n the adjustment of
nitrogen intake to the output l e v e l . This l ag was necessitat
ed by the time involved i n c o l l e c t i o n and analysis of the urine
samples and the c a l c u l a t i o n of the nitrogen content therein.
Towards the close of the experiment d i f f i c u l t y was encountered
i n maintaining a s u f f i c i e n t l y high t o t a l feed intake and fresh
l i v e r was substituted f o r the spray-dried l i v e r meal previously
used, as a protein source. Addition of t h i s fresh product mark-
edly increased p a l a t a b i l i t y of the r a t i o n , r e s u l t i n g i n weight
increases i n the two larger animals and decreased losses In
; the others. The problems of formulation of rations that were
at the same time chemically and physioally suitable were most
d i f f i c u l t and sometimes involved departures from the planned
techniques as i l l u s t r a t e d i n the example above.
Conditions approaching nitrogen equilibrium were attained
i n mink numbers 1, Sa, 3a and 4. The other two animals contin
u a l l y refused portions of t h e i r experimental rations with the
r e s u l t that they evidenced steady declines i n body weight and
corresponding increases i n urinary nitrogen excretion. I t
w i l l be noticed (table IV A) that nitrogen was f i r s t added to
- 44 -
the basal r a t i o n at three l e v e l s corresponding to one, two and
three grams of dried l i v e r meal d a i l y . (See appendix I I f o r
d e t a i l s regarding analyses of r a t i o n constituents.) Nitrogen
equilibrium between feed intake and urinary output was c l o s e l y
approached by one animal at the 103 mg. l e v e l of nitrogen per
animal per feeding and by three others at the 205 mg. l e v e l as
i l l u s t r a t e d i n table IV B.
TABLE IV B: URINARY NITROGEN BALANCE WITH DRIED LIVER MEAL.
Animal No. 1 2a 4-
Exp. Days 29-32 25 T27 30-32 18-26
liean N Intake (Feed) 205 mg. 103 mg. 198 mg. 205 mg.
Sdean N Loss (Urine) 218 mg. 95 mg. 210 mg. 181 mg.
N Balance -13 mg. +8 mg. -12 mg. f 24 mg.
I t w i l l be noticed that mean figures f o r nitrogen intake
and output over several days are quoted rather than the i n d i
v idual d a i l y values i n view of the considerable d a i l y f l u c t u a
t i o n . Only those days showing a reasonably close approximation
to nitrogen equilibrium were considered. One s p e c i f i c instance
of urinary nitrogen equilibrium was also noticed during the
period i n which the animals received fresh l i v e r as t h e i r only
nitrogen supplement. This case i s outlined i n a s i m i l a r manner
to those above i n table IV C. TABLE IV C: URINARY NITROGEN BALANCE WITH FRESH LIVER. Animal No. 2a Exp. Days 31-33
Mean N Intake (Feed) 191 mg.
Mean N Loss (Urine) 177 mg.
N. Balance +14 mg.
- 45 -
I t would appear that a temporary urinary nitrogen e q u i l i
brium can be reached i n the adult mink by i n c l u s i o n of as
l i t t l e as 103 mg. of nitrogen i n the form of dried l i v e r meal
or 191 mg. of nitrogen i n the form of fresh l i v e r i n the d i e t .
Further, a reasonably stable equilibrium can be maintained by
the use of. ,205 mg. of nitrogen i n the form of dried l i v e r
meal. These figures may be translated to represent protein
supplements of 644 and 1289 mg. i n the case of the dried pro
duct and 1194 mg. i n the case of fresh l i v e r . Moreover, bas
ing calculations upon the Kjeldah! nitrogen determinations
car r i e d out on these products i n the Animal N u t r i t i o n Labora
tory, (see Appendix II) one may state that urinary nitrogen
equilibrium can be reached with these animals by the feeding
of 1 gram d a i l y of dried l i v e r meal or 10 grams of fresh l i v e r
and that t h i s condition may be maintained by the feeding of
2 grams of dried l i v e r meal with some suitable non-protein
energy source.
Some words of explanation are necessary at t h i s point
regarding the use of the term "urinary nitrogen equilibrium."
Normally, nitrogen balance experimentation implies a compari
son of nitrogen intake with nitrogen output v i a a l l routes i n
cluding faeces, h a i r and skin losses and the l i k e . In the
present work t h i s concept was recognized at the outset but
recognition was tempered by the a n t i c i p a t i o n of the d i f f i c u l
t i e s involved i n metabolic studies with a "new" animal. Con
sequently, the writer decided, somewhat on the p r i n c i p l e that,
"half a l o a f i s better than no bread," to r e s t r i c t the analy
t i c a l phases of the i n v e s t i g a t i o n to the urinary portion of
FIGURE 4 ( B )
N I T R O G E N B A L A N C E T R I A L D A T A : T O T A L U R I N A R Y N I T R O G E N E X C R E T I O N
K E Y T O R A T I O N S ^
( 2 A . B L = B A S A L + L I V E R 3 A .
' J 3 L M = B A S A L * D R I E D
L I V E R M E A L .
B A S B A S A L ( N F R E E )
0 2 4 6 8 1 0 12 1 4 16 18 2 0 2 2 2 4 2 6 2 8 3 0 3 2 3 4 3 6
T I M E I N D A Y S
8 0 0
7 0 0
6 0 0
5 0 0
4 0 0
3 0 0
2 0 0
- 46 -
the excreta. This method has c e r t a i n d e f i n i t e advantages es
p e c i a l l y i n a preliminary course of experimentation such as
t h i s one because the urinary excretions allow f o r greater
speed coupled with accuracy i n both c o l l e c t i o n and analysis
than do the t o t a l excreta. The writer f e e l s j u s t i f i e d i n
assuming a high degree of d i g e s t i b i l i t y i n the p u r i f i e d rations
used, (see Appendix II) therefore i t i s l o g i c a l to assume that
the major proportion of the animals nitrogen losses would
occur through the kidneys and appear i n the urine.
Ashworth (1933a) i n studies of nitrogen metabolism of
other species stated that the f a e c a l nitrogen on a nitrogen-
free d i e t was nearly constantly 20$ of the t o t a l nitrogen ex
cre t i o n . Morrison (1949) l i s t s the d i g e s t i b i l i t y of commer
c i a l dried l i v e r meal as 96.7$. I t seems probable,therefore,
that even allowing f o r a d i g e s t i b i l i t y c o e f f i c i e n t of as low
as 90$,which i s u n l i k e l y , the f a e c a l nitrogen should not con
t a i n more than 25$ of the t o t a l nitrogen excretion. I t i s
now possible, by use of these assumptions, to estimate the
t o t a l nitrogen l o s s that must be met i n order to e f f e c t a
minimal equilibrium. Increase of 25$ to allow f o r faecal
nitrogen losses would indicate probable establishment of n i t r o
gen equilibrium as l i s t e d i n table IV D. In t h i s table the
nitrogen requirements have been translated into the more
ea s i l y applicable protein complements, always bearing i n mind
that the s p e c i f i c protein i n t h i s case i s supplied by dried
l i v e r meal.
- 47 -
TABLE IV D: TOTAL NITROGEN BALANCE WITH DRIED LIVER MEAL.
Animal No. 1 8A 3A 4
Experimental Days 85-30 85-30 85-30 85-30
Mean Urinary N Loss 178 mg. 183 mg, 801 mg 177 mg
Mean Toal N Loss (calculated
837 " 164 » 868 " 2S6 n
Theoretical Protein Loss 1,481 " 1,085 » 1,675 » 1,475
L i v e r Meal Equivalent 8,314 " 1,601 " 8,617 " 8,306 tt
Actual L i v e r Meal Fed (mean)
8,400 " 1,166 " 1,166 " 8,900
S i m i l a r periods have been studied f o r the various animals
i n order to give a comparative p i c t u r e . I t would appear on the
basis of the mean of the i n d i v i d u a l animals studied that n i t r o
gen equilibrium should be established by the i n c l u s i o n of 1414
mg. of actual protein from l i v e r meal or 8809 mg. of l i v e r
meal i n the d a i l y r a t i o n .
Another i n t e r e s t i n g observation i s afforded by comparison
of the t o t a l l o s s i n body weight of experimental animals with
the losses recorded i n nitrogenous material. Using as samples
animals 1 and 4 f o r which the longest continuous records are
available, t h i s comparison i s presented i n table IV E.
TABLE IV E: PROTEIN LOSSES CO] MPARED TO TOTAL WEIGHT LOSS
Animal No. 1 4 Experimental Days Total Weight Loss
3-87 244 gm.
8-87 390 gm.
[Jrinary N Loss Total N Loss (calculated) Food N (subtract)
5.15 gm. 6.86 gm. 3.88 gm.
5.78 gm. 7.63 gm. 2.67 gm.
Corrected N Loss Protein Loss (dry wt. N x 6.85 Protein Loss (as tissue) % of Total Loss
3.58 gm. )88.37 gm. 31.16 gm. 18.7 <fo
4.96 gm. 31.00 gm. 41.33 gm. 10.6 io
60 jjj
4 0 J—
.02 0 3 0 4 .05 .06 J07 0 8 0 9
C O N C E N T R A T I O N '• M G . C R E A T I N I N E I N S A M P L E
RGURE 4 ( C )
S T A N D A R D C U R V E F O R C R E A T I N I N E
( A L K A L I N E P I C R A T E M E T H O D • F O L I N W J A F F J L R E A C T I O N )
- 48 -
The dry weight of protein l o s t has been converted to re
present tissue protein on the basis of a 25$ dry matter content
determined i n horse muscle f l e s h i n the Animal N u t r i t i o n Labora
tory. In these animals then an average of 11.6$ of the body
weight losses sustained were i n the form of tissue protein.
Variation between the two animals i s probably due to the higher
condition of the heavier mink at the s t a r t of the experiment,
meaning that a correspondingly greater proportion of i t s weight
losses would be i n the form of f a t or water.
Creatinine Excretion
Estimation of creatinine excretion was attempted with two
main purposes i n view. F i r s t , i t was thought that a comparison
of the creatinine excretion of the mink with that tabled f o r
other species i n the l i t e r a t u r e might y i e l d a valuable insight
to the phy s i o l o g i c a l nature of th i s animal. Second, by compari
son of creatinine nitrogen and t o t a l nitrogen excretion data,
an estimate of the b i o l o g i c a l value of the protein supplements
used might well be reached. The general data regarding crea
t i n i n e excretion are presented i n table V.
TABLE V: CREATININE EXCRETION IN MINK ON TEST Animal No, 1 2. 2A 3, 3A 4
Day.Ration Weight Gns.
Urin.N , Mg.
Creat Mg.
Wt. Gms
Jrin. »NMg,
Creat. Mg.
IflTt.T 3ns
Trini Mg.
Creat Mg.
WV Gns:
MB. ?ieat. "Mg.
2 1 F 8 B
10 F 15 BLM 27 BLM 33 BL 35 BL
1035 1005 920 808 761 810 823
'844 310 178 392 200 540 L140
16.32 16.51 14.58 13.74 12.17 12,77 13.09
790 755 675 560
154 89
189 121
12.1C 12.IE 10.36 8.4C
789 740 662 520
406 218 312 539
1144 11*32 9.66 a 32
L42E 138C 12 7E 1172
780 352 498 320 150 532 855
19.13 19.31 17.83 15.74 13. 52 13.70 13.43
2 1 F 8 B
10 F 15 BLM 27 BLM 33 BL 35 BL
1035 1005 920 808 761 810 823
'844 310 178 392 200 540 L140
16.32 16.51 14.58 13.74 12.17 12,77 13.09
60S 573 563
98 83
• 803
9.1? 8.7S 8.61
48* 442 430
187 156 780
6.98 6.42 6JB3
990 99€
100C
780 352 498 320 150 532 855
19.13 19.31 17.83 15.74 13. 52 13.70 13.43
Average 880 515 14.17 580 328 8.86 452 374 6.75 1034 498 16. 09
Rations: F-Fast, B-Basal, BLM-Basal,Liver Meal, BL-Basal, L i v e r .
- 49 -
An examination of these figures (table V, see also figure
4A) leaves one with the impression that there i s l i t t l e ab
normality i n the nitrogen metabolism of mink, at l e a s t as f a r
as may be demonstrated by t h e i r creatinine excretion. In
general, the data obtained i n t h i s experiment f u l l y confirm the
c l a s s i c a l theory of creatinine excretion. Variations are per
ceptible i n the d a i l y excretion of creatinine but i t i s s i g n i
f i c a n t to record that such variations do not correspond to
changes i n protein intake. On the other hand, the l e v e l of
creatinine i n the urine follows c l o s e l y any change i n body
weight or perhaps,more aptly stated, i n muscle mass. (See
figure 4A). Exceptions from t h i s general trend appear i n the
cases of animals 2 and 3 where a rather sharp r i s e i n c r e a t i
nine excretion appears i n conjunction with a prolonged drop i n
body weight. I t w i l l be r e c a l l e d that both these animals died
as a r e s u l t of experimentation and there seems l i t t l e doubt
that the f i n a l r i s e i n creatinine represented the "premortal
r i s e " noted i n other species. Post mortem examination of
these animals presented a picture of severe emaciation and
wasting of muscle t i s s u e , (see appendix I I ) , thus bearing out
t h i s theory.
Following the indications that creatinine excretion may
be most properly evaluated as a function of body weight, the
figures i n table V show the average creatinine excretion for.
the mink to be 15.58 mg. per kilogram of body weight. I t i s
i n t e r e s t i n g to compare t h i s figure to the "Prediction Table"
calculated by Brody, (1945) from his observations on numerous
- 50 -
species wherein he c i t e s a creatinine excretion of 13.2 mg. per
kilogram f o r a 700 gram animal. The accuracy of Brody's predic
tions appears to be borne out once again by the close p r o x i
mity of h i s estimate to the actual a n a l y t i c a l data. A reason
for the s l i g h t increase i n creatinine excretion i n mink above
the expected i s a matter f o r conjecture, however, a strong
p o s s i b i l i t y i s indicated i n the extreme a c t i v i t y , involving
intense muscular action so prevalent i n these animals. One
other possible cause fo r v a r i a t i o n e x i s t s , namely, the occur
rence i n the urine of mink of some stable colouring material
which might a f f e c t the colorimeter readings i n creatinine de
terminations. The reasonable range of creatinine excretion
arrived upon i n t h i s experiment would tend to discount such
a p o s s i b i l i t y yet i t cannot be ignored u n t i l f u rther experi
mental evidence on the subject has been gained.
Information i s also forthcoming regarding the e f f i c i e n c y
of use by mink of l i v e r and l i v e r meal long regarded as s a t i s
factory protein supplements f o r these animals. In the l i g h t
of the data presented i n table V, i t would appear that c r e a t i
nine contains an average of 5.9$ of the t o t a l urinary nitrogen
on the l i v e r meal r a t i o n and 1.7% of the t o t a l urinary nitrogen
on the fresh l i v e r supplement. The p r i n c i p l e involved here i s ,
i n b r i e f , that creatinine as a t y p i c a l endogenous urinary con
stit u e n t , w i l l vary inversely as a proportion of the t o t a l
urinary nitrogen as the t o t a l changes i n conjunction with
changes i n dietary protein. In other words, creatinine n i t r o
gen w i l l be a r e l a t i v e l y large proportion of the t o t a l urinary
- 51 -
nitrogen when a protein of high b i o l o g i c a l value i s fed. The
figures c i t e d herein are merely r e l a t i v e and indicate a some
what greater net e f f i c i e n c y f o r dried l i v e r meal than from the
fresh l i v e r as would be expected from the very physical nature
of the materials. The p o s s i b i l i t y suggests i t s e l f , however,
that continuous and systematic studies of creatinine and t o t a l
urinary nitrogen excretion might lead to rapid and accurate
assessment of the value of i n d i v i d u a l proteins to s p e c i f i c
animals. This concept has been advanced i n the f i e l d of human
n u t r i t i o n by Murlin (1948) and i s presently undergoing further
inves t i g a t i o n i n h i s laboratory at Rochester.
Summary
At the time of i n i t i a t i o n of t h i s study, l i t t l e or no
v a l i d information was available dealing with the nitrogen re
quirements of the mink. On the basis of p r a c t i c a l feeding ex
perience i t appeared that a d a i l y c a l o r i c intake of the order
of 200 - 300 ca l o r i e s per kilogram of body weight would permit
normal maintenance. The protein intake on a "normal" ranch
r a t i o n varies tremendously and i t Is d i f f i c u l t to quote figures
of any tangible meaning.
On the other hand, i f one assumes that the mink f i t s the
normal animal curve produced i n the studies of Ormsby, Benedict,
Brody and others, one might predict a basal d a i l y requirement
of 40 - 80 calories per kilogram and a maintenance requirement
of approximately double these f i g u r e s . In a l i k e manner, one
would expect an endogenous urinary nitrogen excretion of 2 mg.
per c a l o r i e of basal heat which In numerical terms represents
- 52 -
80 - 160 mg. of endogenous urinary nitrogen per kilogram of body weight.
With this experience for guidance, the present work has attempted to confirm or refute these generalities with respect to mink and to determine whether the apparently high level of feeding commonly practised is justified by the metabolic behaviour of the animals. For purposes of clarity and brevity, the results of the present investigation can best be summarized as follows:
1. The difficulties involved in design and operation of equipment for nutritional research with a "new" animal should not be minimized. Points whi3h received special consideration in the present work are listed hereunder:
(a) Caging.- Special cages were constructed to allow for collection of urinary excretions with mink. These cages had of necessity to be escape-proof, and of a size to restrict yet not cramp the normal movements of the animals. Details of their construction are given in appendix II.
(b) Feeding. Considerable experimentation was necessary in order to arrive upon a practical yet accurate method of feeding. Experimental rations and purified diets used successfully with other animals proved unsatisfactory with mink. Certain modifications in existing rations were made and workable mixtures as noted in appendix II were adopted.
(c) Watering. The animals on experiment had to be
- 53 -
provided with s p e c i a l l y designed watering de
vices i n order to ensure adequate supply while
at the same time avoiding s p i l l a g e and consequent
change of urine volume. A closed water b o t t l e
with drinking tube proved most s a t i s f a c t o r y pro
vided a short conditioning period was given the
animals immediately p r i o r to the t e s t ,
(d) Urine C o l l e c t i o n . Stainless s t e e l funnels and
strainers were adopted a f t e r a great deal of ex
perimentation. This material proved admirable
f o r the purpose as i t allowed f o r accurate t o t a l
urine c o l l e c t i o n , and rapid and complete cleaning.
The writer f e e l s that the equipment and method f i n a l l y
evolved i s suitable both from a p r a c t i c a l and a s c i e n t i f i c
viewpoint f o r work with mink. A new animal i s thus available
f o r laboratory experimentation and may lend i t s i n d i v i d u a l pe
c u l i a r i t i e s to the task of expanding the ever-increasing know
ledge i n animal n u t r i t i o n .
2. Endogenous urinary nitrogen excretion with mink has
been shown to follow the general trends exhibited by other
species. This observation i s important i n i t s e l f i n that i t
tends to remove the mink from the sphere of an "unknown" and
"abnormal" animal and place i t instead i n the ranks of those
f o r which constant predictions may be made contingent on the
accumulation of s c i e n t i f i c data. Actual endogenous urinary
nitrogen excretion of the two animals tested averaged 565 mg.
per kilogram of body weight d a i l y . Using the commonly accepted
- 54 -
r a t i o of 1 c a l o r i e of basal heat to each 2 mg. of endogenous
nitrogen, t h i s would indicate a B.M.R. of 287 calories per
k i l o or approximately 200 c a l o r i e s f o r a 700 gm. animal. I t
would appear that a higher rate of heat production e x i s t s i n
the mink than might be expected from the general "prediction"
tables. (Brody, 1945) The causes of t h i s additional c a l o r i c
increment are probably the extreme nervous temperament and
high state of muscular a c t i v i t y exhibited by these animals
and even under the c a r e f u l l y regulated experimental conditions
a quiescent state was not attained.
3. Nitrogen Balance. Conditions of nitrogen equilibrium
were attained by the i n c l u s i o n of 1414 mg, of actual protein
i n the d a i l y r a t i o n of a 726 gm. mink, that i s 1947 mg. of
protein per kilogram of body weight. In t h i s experiment as i n
the one outlined i n part (2) above, the condition of the t e s t
animals was c l o s e r to what i s known as the "maintenance" stan
dard than to a true basal l e v e l . On the basis of the experi
mental evidence gathered herein, i t would appear that an
average sized female mink might be maintained i n nitrogen
balance on a d a i l y dietary supplying s l i g h t l y l e s s than 2 grams
of actual protein. I t must be emphasized once again that n i t r o
gen balance experiment figures are v a l i d only insofar as the
s p e c i f i c protein employed i n feeding i s concerned and hence
the figures cited above must be applied i n terms of l i v e r meal
protein.
4. Creatinine Excretion. The results obtained following
investigations into the rate of creatinine excretion of mink
- 55 -
were most i n t e r e s t i n g . As with other species, creatinine ex
cretion by minlr proved to be extremely constant and appeared
to vary d i r e c t l y with body weight. No evidence was discovered
to refute the theory that creatinine excretion i s l i t t l e af
fected by dietary nitrogen content. An average rate of crea
t i n i n e excretion of 15.58 mg. per kilogram of body weight was
established f o r the animals under in v e s t i g a t i o n as compared to
a predicted excretion from the l i t e r a t u r e of 13.2 mg. per k i l o
gram f o r an animal of s i m i l a r s i z e . The variance between ac^
tual and predicted values f o r creatinine excretion i s thought
to be due to the intense muscular a c t i v i t y of the mink.
5. P r a c t i c a l Implications. While i t i s hoped that some
small contribution to the s c i e n t i f i c knowledge of n u t r i t i o n
has been made by t h i s present work, the writer f e e l s that the
p r a c t i c a l applications from such knowledge, duly confirmed,
could be most extensive. Without delving into d e t a i l , t h i s
work would appear to indicate that the mink may shortly be
subjected to r i g i d feeding standards i n common with other
domestic animals. The suggestion i s put forward that very
considerable overfeeding of protein has been indulged i n with
these animals, probably on the premise that, as carnivora,
they require diets r i c h i n muscle f l e s h . The experiments
ci t e d herein indicate very d e f i n i t e l y that a nitrogen e q u i l i
brium may be maintained through the administration of extreme
l y small quantities of protein and a presumption may be made
that t h i s protein need not a l l be of animal o r i g i n provided
that the needs f o r e s s e n t i a l amino acids are met. One might
- 56 -
expand the theme ad infinitum yet l e t i t s u f f i c e to say that
provided a sound basis i s b u i l t upon s c i e n t i f i c f a c t s , there
i s no reason why the n u t r i t i o n a l requirements of mink may not
be reduced to numerical terms and established as a matter of
common knowledge.
6. Recommendations and Suggestions. C r i t i c i s m may be
made of the present work on the grounds that the numbers of
animals involved are i n s i g n i f i c a n t . The reason f o r t h i s pau
c i t y of numbers i s simple In that the time involved i n devis
ing equipment and experimental methods was so considerable that
further experimentation became impossible. Moreover, the mere
care and maintenance involved i n operation of the stock colony
of animals was also extremely time consuming and yet t h i s
labour v/as b a s i c a l l y necessary f o r the whole conduct of the
experiments. The writer would urge that the preliminary i n
sight into the various requirements of mink, as contained i n
th i s t h e sis, be exploited i n fur t h e r investigations so that
the actual time available f o r research may be u t i l i z e d to the
f u l l .
Further i n v e s t i g a t i o n appears indicated i n the f i e l d s of
nitrogen and energy balance, b i o l o g i c a l values (possibly as
demonstrated through creatinine studies) and d i g e s t i b i l i t y of
d i f f e r e n t i n d i v i d u a l nutrients, and feed mixtures. I t i s only
through c o r r e l a t i o n of information gained by means of these
devious methods that a sound basis for the n u t r i t i o n of mink
may be f i n a l l y approached.
APPENDICES
The following relevant data
are included i n Appendix
form f o r reasons of
spacing and
arrangement.
- i -
APPENDIX I: PREPARATION OF REAGENTS AND LABORATORY TECHNIQUES.
1, Standard Acid Preparation: (Chemical Rubber Pub. Co., 1948)
St a r t i n g with HC1 of density about 1.10, constant b o i l i n g HC1 i s prepared by d i s t i l l a t i o n and discard of the f i r s t f of the l i q u i d passing over. Correction must be made f o r v a r i a tions i n atmospheric pressure. The following figures are suggested by Hollingsworth and Foulk:
Barometric Pressure %HC1 by Weight Wt. HC1 f o r IN Solution
770 20.197 180.407 gm. 760 20.221 180.193 750 20.245 179.979 740 20.269 179.766 730 20.293 179.555
The amount of acid needed i s weighed out accurately, using a c a p i l l a r y or Pasteur type pipette to f i n i s h and i s dil u t e d to the required volume. For example: making 4 l i t r e s of N/14 acid from constant b o i l i n g HC1 at 770 l b s . pressure, use:
180.407 x 4 . 51.5448 gm. 14
2. Standard A l k a l i Preparation: (Hawk, 1947a)
a. Preparation of carbonate-free NaOH: Shake up 110 gm. best quality NaOH with 100 gm. d i s t i l l
ed water i n a 300 ml. Erlenmeyer f l a s k to make a saturated .sol'n. Stopper, and allow to stand u n t i l the sodium carbonate s e t t l e s to the bottom leaving a layer of clear, saturated NaOH sol'n. p r a c t i c a l l y free from carbonate.
b. Preparation of a Standard NaOH solution: For 4 l i t r e s of standard N/14 solution, measure out
17.96 ml. of the saturated NaOH sol'n. into a large f l a s k , (6 l i t r e Erlenmeyer) add 3000 ml. d i s t i l l e d water and mix thoroughly. Rinse a clean burette with the a l k a l i sol'n. prepared, f i l l , and t i t r a t e the sol'n. against the standard N/14 acid prepared as above, using 1% a l c o h o l i c phenolphthalien as indicator.
Calculate the normality of the a l k a l i sol'n. from the t i t r a t i o n and d i l u t e u n t i l a N/14 sol'n. i s obtained. At a l l times shake the sol'n. thoroughly to ensure thorough mixing. Store the a l k a l i i n a stoppered, p a r a f f i n - l i n e d b o t t l e . A 4 l i t r e aspirator bottle i s convenient f o r use.
3. Mixed Indicator Preparation: (Zuazaga, 1942)
Prepare a 6.1% sol'n. of Bromcresol Green. Prepare separately a 0.1% sol'n. of Methyl Red i n 95% alcohol. Mix the two indicators i n the proportion of 5 parts Bromcresol Green to 1 part Methyl Red sol'n.
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4. Kjeldahl/Gunning Method for Nitrogen Determination, (Koch, 1934) with Modifications:
Reagents: a. Standard HC1 and NaoH sol'ns. (N/14) b. Concentrated H2S04. c. C U S O 4 sol'n., 10%. d. K 2 S O 4 , reagent grade. e. Pumice, powdered, Kjeldahl grade. f . NaOH sol'n., 40%. g. Mixed Indicator as above.
Procedure: Pipette accurately a 1 ml. sample of the urine into a
Kjeldahl f l a s k , add 10 ml. H 2 S O 4 , 1 ml. C U S O 4 sol'n. and 5 gm. K 2 S O 4 . Also prepare a blank, using the same amounts of reagents but no urine. Place the f l a s k s on the digestion rack and heat gently, l a t e r intensely, u n t i l the reaction mixture i s a c l e a r , l i g h t green. When digestion i s complete, sw i r l f l a s k s to get contents on walls, d i l u t e with 100 ml. d i s t i l l e d water and set aside to cool.
Measure c a r e f u l l y 50 or 100 ml. (depending on the amount of nitrogen presumed to be i n the sample) of standard N/14. HC1 into 250 ml. Erlenmeyer c o l l e c t i o n f l a s k s . Place these fla s k s under the t i p s of the adaptors on the d i s t i l l a t i o n shelf. Apply the heat on the heating elements to be used i n the d i s t i l l a t i o n . To each Kjeldahl f l a s k containing the digestion mixture, add a generous spoonful of the powdered pumice to prevent bumping and pour evenly and slowly down the side of each f l a s k 40 ml. of the 40% NaOH sol'n. Connect the flasks with the traps on the d i s t i l l a t i o n apparatus and s t e a d i l y rotate them to ensure thorough mixing of the contents. Immedi a t e l y place the c o l l e c t i o n f l a s k s so that the t i p s of the adaptors are beneath the surface of the contents, r a i s e the heat under the d i s t i l l i n g f l a s k s and d i s t i l about ^ the quant i t y over. Remove the c o l l e c t i o n f l a s k s and t i t r a t e the standard acid remaining against the standard a l k a l i , using the mixed indicator previously described.
Calculate the number of milligrams of nitrogen i n the sample. As the standard acid was N/14, each ml. of acid used up represents one mg. of nitrogen i n the form of ammonia. From t h i s f i g u r e , calculate the t o t a l number of mg. of n i t r o gen excreted by the animal during the entire c o l l e c t i o n period.
5. F o l i n - J a f f e Method for Creatinine Determination with Modif i c a t i o n s (Peters, 1942):
a. Preparation of P u r i f i e d P i c r i c Acid: (Hawk, 1947b)
(Ordinary CP P i c r i c Acid forms too deep a colour f o r iaccurate photo-colorimetric procedures.)
Transfer 500 gm. of moist p i c r i c acid to a Florence f l a s k of 1500 ml. capacity. Add 500 ml. acetone and shake with a
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l i t t l e warming under hot tap water u n t i l a l l the c r y s t a l s have dissolved. Add 20 gm. Norit activated charcoal, shake, and f i l t e r into another f l a s k .
Dissolve 250 gm. of anhydrous NagCOg and 100 gm. of NaCl i n 2500 ml. of warm water i n a large "beaker. S t i r slowly with an agate-ware spoon or glass rod and add the acetone sol'n. gradually to the a l k a l i n e s a l t sol'n. When the evolution of C0g has f i n i s h e d , l e t stand i n cold water f o r about % hour, and f i l t e r on a large (20 cm.) Buchner funnel. Wash with 2 l i t r e s of 7% NaCL sol'n., and suck as dry as possible.
Return the ppt. to the beaker and add 2 l i t r e s of b o i l i n g water and 20 gm. of NagCOg. To t h i s hot sol'n. add gradually with s t i r r i n g s 150 gm. of NaCl. Cool, f i l t e r , wash as before with 7% NaCl, then with 2% NaCl and f i n a l l y with methyl alcohol to remove most of the remaining chloride and water. Dry at room temperature.
P i c r i c acid i s prepared from the picr a t e prepared as above by treatment with d i l u t e HC1. Prepare 2 l i t r e s of d i l u t e HC1, (1 v o l . cone. HC1. to 4 v o l . water) and pour the acid over the p i c r a t e , s t i r r i n g with a glass rod to ensure complete act i o n . F i l t e r again through the Buchner funnel, using hardened f i l t e r paper. Dry the p i c r i c acid c r y s t a l s i f they are to be used immediately. Temperatures up to 90°C may be s a f e l y used i n the drying of p i c r i c acid.
b. Estimation of Creatinine i n Urine: (Peters, 1942)
P r i n c i p l e : A tungstic acid f i l t r a t e of urine i s treated with a mixture of p i c r i c acid and sodium hydroxide. A red compound i s formed which, with the yellow of the excess p i c r i c acid, produces an amber coloured sol'n.
Procedure: Transfer 5 ml. urine to a 100 ml. volumetric f l a s k , d i l u t e to volume and mix. Transfer 2 ml. of t h i s d i l u t ed urine to a f l a s k , add 16 ml. of N/12 H 2 S O 4 . Mix. Add 2 ml. 10% Na2W04# Shake. F i l t e r through Whatman no. 40 paper.
Transfer 5 ml. of the f i l t r a t e to an absorption c e l l , and 5 ml. of d i s t i l l e d water to a s i m i l a r c a l l f o r a blank. To each add 2.5 ml. of fresh a l k a l i n e p i c r a t e . (1 v o l . 10% NaOH to 5 v o l . 1.175% p i c r i c acid. This pic r a t e must be used within 5 minutes.) Mix thoroughly.
Let the mixture stand f o r 20 minutes. Read i n a c o l o r i meter or spectro-photometer, using a wavelength of 520 mu. In practice a standard curve was prepared using a standard creatine sol'n, d i l u t e d over the range expected to be encountered. Measurement was made i n a Coleman spectro-photometer.
5. Detection of albumin i n Urine: (Hawk, 1947c) Place 5 ml. of Robert's reagent (1 v o l . cone H N O 3 and 5
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v o l . saturated mg. S O 4 ) i n an i n c l i n e d t e s t tube and slowly pipette urine down the side of the tube. P r e c i p i t a t e d protein w i l l form a white l a y e r at the interface of the two sol'ns.
6. Detection of B i l e i n Urine: (Hawk, 1947d) Rosenbach's Modification of the Gmelin Reaction.
F i l t e r 5 ml. urine through a small f i l t e r paper. Introduce a drop of cone. H N O 3 at the apex of the paper. Presence of b i l e pigments i s indicated by a succession of d i f f e r e n t colours spreading out from the centre.
7. Detection of Glucose i n Urine: (Hawk, 1947c) Benedict's Test.
a. Preparation of Benedict's Sol'n:
Reagents: Copper sulphate - 17.3 gm. Sodium c i t r a t e - 173.G gm. Sodium carbonate - 100,0 gm. D i s t i l l e d water to make 1 l i t r e .
With heating, dissolve the sodium c i t r a t e and carbonate' i n about 800 ml. water. F i l t e r i n t o a graduate and make up to .850 ml. Dissolve the C U S O 4 i n 100 ml. water and add i t . s l o w l y to the citrate/carbonate sol'n. with constant s t i r r i n g . Make up to 1 l i t r e .
b. Benedict's Test:
To 5 ml. of Benedict's reagent prepared as above, add exactly 8 drops of urine. B o i l the mixture vigorously f o r 2 minutes, then allow to cool spontaneously. Presence of glucose iH indicated by a heavy curdy ppt. of varying colours; apple green, yellow, or red, depending upon the amount of sugar present.
8. General Laboratory Procedure:
Analyses of urine f o r t o t a l nitrogen were made d a i l y , and i n duplicate. The mean value was taken as the actual nitrogen content. Creatinine determinations were carr i e d out on samples of urine collected on various predetermined dates throughout the experiment. Representative samples were taken during the pre-test, f a s t i n g , nitrogen-free, and complete synthetic d i e t periods. Repeat analyses were, of course, c a r r i e d out wherever close agreement was not attained i n analysis of the duplicates. In such cases where urine samples had to be kept overnight, they were placed i n a r e f r i g e r a t o r and under an overlay of toluene•
9. Investigation re Contamination of Urine Samples:
I t was noticed that mink undergoing nitrogen balance determinations shed considerable h a i r , underfur and skin debris, es p e c i a l l y during the spring and early summer months. In an
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, attempt to determine whether the N content of the urine would a l t e r i n passage over t h i s matter, the following experiment was i n i t i a t e d :
a. The amount of h a i r shed hy one mink i n a day (including skin brushings and anything other than faeces and urine) was col l e c t e d and was found to weigh 0.2490 gm.
b. A 40 ml. sample of urine (about an average day's excretion) was c o l l e c t e d , an ali q u o t analyzed f o r t o t a l N by the Kjeldahl method and the remainder poured over the above h a i r sample i n a 125 ml. Erlenmeyer f l a s k and allowed to stand f o r 24 hours.
c. A representative sample was taken from t h i s "extracted" urine, f i l t e r e d , and analyzed f o r t o t a l N as previously. The res u l t s (mean of two determinations) are given below:
Total Nitrogen (mg. per ml.) Sample
10.0 mg. 9.8 mg.
Straight Urine Urine/Hair Extraction
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APPENDIX I I : ANIMAL TECHNIQUES
1. Housing and Care of Animals
Cages were devised that would ensure a reasonable amount of space f o r the animals while at the same time r e s t r i c t t h e i r a c t i v i t y towards a basal l e v e l . These cages had to be designed to allow f o r complete c o l l e c t i o n of a l l excreta. The oages themselves were fabricated from squared wire fencing and measured 18" i n height and s l i g h t l y l e s s than 20" i n diameter. Each cage was equipped with a s l i d i n g door to which feed trays could be attached and a 150 ml. water bot t l e with drinking tube. Animals were removed f o r examination or weighing by means of a box trap.
Urine c o l l e c t i o n was c a r r i e d out by means of 20" diameter stainless s t e e l funnels placed immediately under the cages. These funnels were constructed with a short v e r t i c a l rim to ensure complete c o l l e c t i o n of excreta. Faeces were separated from the urine by means of two st a i n l e s s s t e e l wire mesh screens placed i n each funnel. Hair and skin debris was separated out by a l i g h t l y packed glass wool f i l t e r placed i n the narrow neck of each funnel. The actual urine containers were 100 ml. graduated cylinders hung d i r e c t l y under the funnels on pierced rubber stoppers. Toluene was used to exclude a i r from the urine samples during c o l l e c t i o n . With the exception of the funnels and screens, a l l equipment was constructed and assembled by the writer at the Animal N u t r i t i o n Laboratory.
Immediately a f t e r measurement of the t o t a l d a i l y urinary excretion, samples were transferred to te s t tubes and kept i n a r e f r i g e r a t o r under toluene u n t i l analysis could be c a r r i e d out. A breakdown diagram of equipment used and a photograph of the u n i t i n operation are included as Figures 5 and 6.
- W I R E M E S H C A G E ( 2 0 " D I A . , 718" H T , T ' S Q U A R E S )
- W A T E R B O T T L E
" S P R I N G C L I P
- C A G E D O O R ^ L I D I N G
- S T E E L A N I M A L G U A R D
• D R I N K I N G T U B E
S T A I N L E S S S T E E L F U N N E L
W I R E M E S H F A E C E S S C R E E N
W I R E S C R E E N G L A S S W O O L F I L T E R
G R A D U A T E D C Y L I N D E R
F I G U R E 5 • D I A G R A M O F A N I M A L E Q U I P M E N T A D O P T E D
F I G U R E 6 « P H O T O G R A P H O F U N I T I N O P E R A T I O N
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2. Experimental Ration Preparation
(a) Nitrogen-free energy source. Two nitrogen-free diets were compounded as l i s t e d below:
i . Starch d i e t (used i n preliminary experiment) (Ashworth, 1933).
Corn Starch - 74.0 gms. Lard - 8.0 " Cod L i v e r O i l - 2.0 » Sucrose - 10,0 " S a l t Mix, U.S.P. II - 4.0 " Cellulose - 2.0 "
The cod l i v e r o i l used was a standardized product having a potency of 43,780 I.U. vitamin A per gm.
Cellulose was provided by shredding the dry weight required of Whatman no, 1. f i l t e r papers and pulping them i n a known weight of d i s t i l l e d water i n a Waring Blendor. This r a t i o n supplies approximately 4.2 c a l o r i e s per gram.
i i . Sucrose d i e t (used i n the second experiments) (Frost, 1946)
Sucrose 73 gms. Lard 20 « Corn O i l 3 i t Cod L i v e r O i l - 0.5 t i S a l t Mix (U.S.P. II) - 4.0 i t Choline Chloride 0.1 i t Agar 1.0 n Thiamin HC1 0.6 mg. R i b o f l a v i n 0.6 t t Nicotinamide 12.0 II
Pyridoxine HC1 0.4 t i Ca. Pantothenate 1.2 t i
I t w i l l be noticed that t h i s r a t i o n was strongly f o r t i f i e d with the B vitamins i n an e f f o r t to combat anorexia which normally occurred during long periods of nitrogen-free feeding. The cod l i v e r o i l used was a high-potency standardized product containing 43,780 I.U. vitamin A per gram. This d i e t supplies 5,1 c a l o r i e s per gram and was fed at a rate ensuring 200 calories per kilogram body weight.
Kjeldahl nitrogen analysis of the sucrose d i e t indicated a mean nitrogen content of 0,025%,
(b) Nitrogen sources.
The nitrogen source o r i g i n a l l y planned f o r t h i s experiment was a standardized spray dried l i v e r meal produced by the Valentine Meat-Juice Co., Richmond, Va., U.S.A. K j i l d a h l
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determinations on t h i s product showed a mean nitrogen content of 10.27%* The advantages of such a supplement were obvious -i t offered a uniform, powdered nitrogenous food which could be e a s i l y stored and accurately weighed i n small quantities. Certain disadvantages i n e i t h e r p a l a t a b i l i t y or physical texture soon became apparent, however, and low t o t a l r a t i o n consumption l e d to the use of fresh hog l i v e r as a nitrogen source.
The fresh l i v e r used was a.product of Canada Packers Ltd., branded as "hog l i v e r , inedible, f o r animal food only." The l i v e r was kept frozen u n t i l s h o r t l y before use and representat i v e samples were taken, avoiding the outer surfaces which might-have become dehydrated. For convenience i n weighing, the l i v e r samples were f i n e l y chopped i n the frozen condition e
thus avoiding l o s s of moisture or blood. Mean of Kjeldahl determinations carried out on the fresh l i v e r showed a nitrogen content of 1.91% on a wet weight b a s i s . Dry matter content was established as 32.5%.
3. Administration of Rations
A great number of t r i a l s were necessary before a s a t i s factory method of feeding could be devised due to the habit inherent to mink of carrying food from any container before devouring i t .
A most s a t i s f a c t o r y method consisted of mixing the r a t i o n with a known weight of d i s t i l l e d water to a pasty consistency and then expressing the-desired amount of the mixture to the animal through a hard glass tube. In t h i s manner the amount fed could be accurately regulated and there was p r a c t i c a l l y no l o s s . This method proved very s a t i s f a c t o r y with the f i r s t basal r a t i o n but was discarded with the second r a t i o n f o r fear of l o s i n g appreciable amounts of the sucrose i n s o l u t i o n . A box feeder was devised f o r use with the second r a t i o n and s p i l l a g e loss was subtracted from the amounts fed. Diagrams of the feeders used and a photograph of one i n operation are presented as figures 7 and 8.
4. Loss of Experimental Animals
A l l losses of experimental animals Incurred i n both parts of the experiment showed the same general picture, as follows:
i . Ante-mortem examination. The animals appeared normal up to a period of 3 or 4 days before t h e i r death a f t e r which they became l i s t l e s s and s t e a d i l y weaker. When offered feed of any nature, these animals refused i t completely. Samples of urine co l l e c t e d the l a s t two days ante-mortem showed evidence of haematuria and yielded increased nitrogen content figures on analysis.
i i . Post-mortem examination. Autopsy of the animals showed
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them t o he e m a c i a t e d though n o t e n t i r e l y d e v o i d o f m e s e n t e r i c f a t . P e t e c h i a l haemhorrage s p o t s were e v i d e n t on t h e l i v e r and a c e r t a i n amount o f b l o o d and o t h e r f l u i d was found f r e e i n the a b d o m i n a l c a v i t y . S p l e e n s were somewhat e n l a r g e d and k i d n e y s appea red s l i g h t l y p a l e i n c o l o u r . The appea rance o f t h e l u n g s i n one o f t h e s e a n i m a l s was s u c h as t o i n d i c a t e t h a t pneumonia had been a c o n t r i b u t i n g cause o f d e a t h .
F I G U R E 7 E X P E R I M E N T A L F E E D I N G M E T H O D S
I. B O X T Y P E F E E D E R
F I G U R E 8 P H O T O O F 2 ( A B O V E ) I N O P E R A T I O N
APPENDIX II I ADDITIONAL DATA RE MINK NUTRITION
1. Natural Diet of the Mink.
Any n u t r i t i o n a l study of a recently domesticated animal should include some mention of the p a r t i c u l a r animal's d i e t i n the wild state under more or less natural conditions. Such a d i e t should not be adopted as a- r i g i d standard since what we look on as "natural" conditions have undoubtedly been considerably r e s t r i c t e d by the inroads of our modern c i v i l i z a t i o n ; yet the d i e t selected by the animal when i t has any degree of free choice offers a valuable guide to the p a l a t a b i l i ty of various feeds to that animal. Also the i n s t i n c t f o r self-preservation i n the wild animal probably leads i t to the choice of a reasonably balanced diet,therefore,some informat i o n on the n u t r i t i v e requirements f o r the same animal under domestic conditions may possibly be gleaned from t h i s study.
In the case of mink, most studies of the animals' d i e t i n the wild state have been conducted by the Wild L i f e services of countries to which the mink i s native. One of the foremost investigators i n t h i s f i e l d , ( B a i l e y , 1930), i n a study c a r r i e d out i n Yellowstone National Park wrote that the general di e t of wild mink consisted of f i s h , frogs, crustaceans and to some extent, mice, gophers, muskrats, ground s q u i r r e l s , chipmunks, birds and other small game. G r i n e l l (1937) i n his studies of C a l i f o r n i a Wild L i f e carried the examination s t i l l f a rther to include the gross percentage composition of the mink's stomach contents. He reports that laboratory examination of 149 mink stomachs from d i f f e r e n t parts of C a l i f o r n i a repealed the contents to be the following percentages by bulk:
Canadian surveys,(Cowan,-1948), bear out the above f i n d ings i n the main but indicate that the f a r northern type of Yukon mink d i f f e r i n pr e f e r r i n g rodents f o r food wren when f i s h i s r e a d i l y a v a i l a b l e . Observations of the habits of wild mink, (Bailey, 1936), have shown that the animal i s r a r e l y found f a r from water; therefore, i t may be assumed that i n many oases f i s h and other acquatic l i f e constituted a major portion of the d i e t . This same work names crustaceans as being the f a v o r i t e food of wild mink and i n regions where they are abundant the p r i n c i p a l food the year around. According to traces observed i n the droppings of wild mink i n and near t h e i r dens, any game i s consumed p r a c t i c a l l y i n i t s entirety: bones, feathers, f u r , scales, s h e l l s and a l l .
F i s h Birds Small Mammals Crayfish and Mussels Non-Food Material
39.6% 27.0% 21.5% 3.4% 8.5%
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2. Time of Passage
Naturally the use of food materials by d i f f e r e n t species varies considerably according to the time available f o r action of the various digestive mechanisms on that food. A/search of the l i t e r a t u r e revealed no information i n t h i s regar&T^there-fore, a b r i e f experiment was set up to determine time of passage, using animals of the Unive r s i t y Mink Colony as subj e c t s . A reagent grade of Carmine (Merck) was used as an i n dicator and was mixed with a normal stock d i e t i n the quantity of approximately 1 gm. to 150 gms. of the wet r a t i o n . T r i a l s were run i n duplicate on two d i f f e r e n t pairs of animals. The time of feeding was accurately recorded and appearance of the dye i n the faeces was taken as in d i c a t i v e of time of passage of food material. Aotual time of defacation was not recorded i n any case but the several t r i a l s agree on an approximate time within the range of 7 to 9 hours.
3. Weight-Growth Correlations.
I t i s evident that once a maintenance standard has been set for mink n u t r i t i o n , supplementary requirements allowing for the processes of growth, production and reproduction must shortly be arrived upon. In order to assess the rate of growGi i n mink k i t s on a "standard" r a t i o n and hence l a y the basis f o r calculations of growth requirements, four l i t t e r s of mink from the Uni v e r s i t y Mink Colony were weighed weekly from b i r t h with the following r e s u l t s :
*TFfoL€. N \ ^tvCMl ZlMM Cflft(leVM\o^ vA UTTERS of NovlHG, [AvM 0? itortlBUftL 9itfWtok\_S
%, i ^ i
3
fife. \ WKS) i 4 5 c \A ME 1 L (A L s (A
VI WW in si 5f 7? t\ 17 to IA I-41 XX 23 z 47 4r zz • £•57 ft ZS7 us 11 i«7 lOC) 1 0 1 2.1* 51 53 34 74 r7 12 f4 St - m III |0£ lo i $-n. ̂7 lo? MS" let in J44 41 St) <?<? /// "7 /of
15T5" \3"l l£>7 \Ii 4.J7 |W IJo \?) I3t I4\ 154 4 if i n I 0 1 lo7 r /.?sr «7 /4? 11 ~ 5,9) l\\ IbO \S7, IS? IS4 I43 174 |7i III f - V | 141 131 I37J L 14? 1U So » t.D iLt> l \ \ 14? U \ X|f 24o 1 S 1 :rt> lof 141 fe.iT i n \f? 2£>o| 7 Zu SIS 32X
in » 7.0 Si? SOD 256 7S7 S?7 243 3oT 2i4 171 ?.iq wr 17j- Iff f 43c JU 4 J u l . lq 34S 3io 177 Si4 W7 3tr 33f sir 173 34o 342 341 "3 ?77 406 4V 424
These data are untreated and are included f o r reference only. Mean values "M" .are given f o r each l e t t e r at each weighing.
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4. Relation Between Organ Weights and Body Weight of Mature Mink.
Structure and function are two aspects of the same thing. Each s t r u c t u r a l det a i l possesses i t s functional expression....
Carrel
This project has been undertaken f o r the purpose of correl a t i n g organ weights to body weight i n mature mink thus paral l e l i n g s i m i l a r studies already oompleted f o r other animals. In attempting research into the n u t r i t i o n a l requirements of mink, the writer has been hampered by the lack of a v a i l a b l e "normal" data on these animals which might serve as a basis f o r c a l c u l a t i o n s . D i f f i c u l t i e s have been encountered i n the formulation and administration of experimental rations to mink which suggest the p o s s i b i l i t y of physiologic and anatomic d i f ferences between them and other experimental animals. The various body functions with which we are interested, such as energy and protein metabolism, tissue production (growth)," fur production, and reproduction a l l depend f o r t h e i r construct i v e materials upon the e f f i c i e n c y of the organs of the digest i v e t r a c t . A knowledge of the correlations of these various organs to body size may well be a convenient s t a r t i n g point i n a study of t h e i r function although i t must be remembered that changes i n h i s t o l o g i c a l structure (and corresponding physiolog i c a l action) may invalidate relationships based only on s i z e . I t i s hoped that t h i s present study may shed some l i g h t on the functional e f f i c i e n c y of mink and that i n c i d e n t a l l y i t may o f f e r a normal standard against which abnormal conditions may be contrasted and evaluated.
Method.
Weight, being a function of volume, was taken i n preference to length as a standard f o r comparison. Mature animals were chosen as subjects i n order to reduce v a r i a b i l i t y due to age.
The animals chosen f o r the purpose of t h i s experiment were mature mink of both sexes and of the standard or dark type. They were k i l l e d by gassing i n an a i r t i g h t chamber immediately a f t e r which the complete body weight was recorded and the p e l t removed. The number of animals k i l l e d over a short period of time i n order to obtain the s a t i s f a c t o r y "primeness" of p e l t necessitated storage of the carcasses p r i o r to diss e c t i o n . This was effected through sharp-freezing and glazing of the carcasses with water i n order to prevent extreme evaporation.
At the time of dissection, the v i s c e r a were c a r e f u l l y removed, separated where applicable and weighed immediately. Use of a team system whereby ce r t a i n operators were assigned to a balance or to the task of d i s s e c t i o n reduced the exposure time to a minimum. Weights were taken of the carcass with p e l t
- x i i i -
removed, l i v e r , heart, lungs, stomach, i n t e s t i n e , kidneys and spleen. Any excess blood was allowed to drain o f f on to absorbent paper before the organs were weighed and as l i t t l e surrounding f a t as possible was removed with the digestive t r a c t .
From the weights obtained percentages of t o t a l body weight were calculated and the re s u l t s tabulated.
INTEST. WT
Va'W 3.3 4Ut, 3.X 41 fI 3J 44. to J.i
KIDNEYS WT %
/O. /o o. IS 194 0.13
ib.i3 olo
/OC? 07/ 19Z 0.7) 9J9 on fit c& 9-91 0.7S /Oil oi4
7.41 0%
113 6.9 f $:s o is
7.1 6. ft 4f AS1
HEART | LUNGS | STOMACH I S P L E E N
L41 6. X
i ±
73 Uf it 71 72 53 11 7j__U_ 70 ill 4.1 1) l4.v 41 17 n.o Cl lo fo.c f/
14 4ft 4*
71 4Lo <:./
13 VJt 41
_V 91 6J_ tl 4h 63 7/ H 4LL
72 &o I >
14.0 4,4 91 o U
0.93 WB1 o.t4 If. SI oil 11.34 0.IJ
6.13 U3 e.fi
931 Cit TMk OlX
6.74 L47 OS?
I.K 0/4 3.00 0.21 B E 1 H H 2.30 0/1
o/6 J 31 on
9-/f 0 71 9-o oil
94 o%
/24\ 6.71
on 14/ on
WESmWUBSBEL I1l\ 0./f\ //(, O./f /.ill o.n\ nl 0J4
KHHaiEJIlHIHIiaEailH
ti.F ns 94 OlD
jt.x 114 1.1 0.S4
/ir llo ix CS9 ft2C 1.24 llK tjf /i.4 /.21 P.of Q.IS /J3f 1.21 90 0.(2, left >X4 II Oio l i t 1.41 11 biO
J.I c./r 11 o>4 "2 1 t.ii J.I 0/1 IS on n c./4 13 O-li
6.1 c.tx wzm II. if Off EH na 16-SS 2.3 oil at 1.4 0/4 /I.O 01) *4J D.li
34.7 2.0 1.1 Oil So. 1 3.1-x 2-3 0-/C
1i 0.19 2.X on
19-3 2/3 2.0 D.2Z
1/4 Oil 13 M r n.9 0.11 4.0 6.24 114 /if 2.x o i4 ll4 m 3.4 t /9
ff.4 0 9? 2.1 til Oil / 3 O./i
xiv
5 . Basal Metabolism Data for Mink During the course of experimentation, values for the
basal metabolism of the mink were calculated from urinary nitrogen excretion. In order £ 0 check the accuracy of these figures, actual oxygen consumption of the last three test animals was measured over definite periods of time, using a Mc Donald College respirometer apparatus. A certain amount of d i f f i cu l ty was encountered in maintaining a quiescent state i n the animals after a preliminary 24 hour fast, •however conditions reasonably near basal were attained. The data obtained are l i s t ed as follows:
OXYGEN CONSUMPTION -IN MINK 23 JULY, 194-9
'Wol Weight 02/min. O2/24 hrs. BMR c a l . Cal/Kg Tests gm. cc. calculated calculated calculated
4 1000.0 16.8 23.19 1. 111.33 111.33 6
1 83O.O 19.2 27.65 132.74 159.93 6
2A 560.0 20.3 29.23 140.33 250.59 6
The thermal equivalent per l i t r e of oxygen was taken to be 4.801 calories, corresponding to an R.Q. of 0.8, i n accordance with figures cited by Brody, "Bioenergetics and Growth", p. 310.
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