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

- i v -

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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