THE AMINO ACID CONTENT AND NUTRITIVE VALUE
OF THE PROTEINS OF COTTONSEED MEAL.

BY

WILLIAM BARBOUR NEVENS

B.S. University of Wisconsin, 1914

THESIS

Submitted in Partial Fulfillment of the Requirements for the

Degree of

DOCTOR OF PHILOSOPHY

IN ANIMAL HUSBANDRY ~

IN
THE GRADUATE SCHOOL

OF THE

UNIVERSITY OF ILLINOIS
1921

THE AMINO ACID CONTENT AND NUTRITIVE VALUE
OF THE PROTEINS OF COTTONSEED MEAL

BY

WILLIAM BARBOUR NEVENS

B.S. University of Wisconsin, 1914

THESIS

Submitted in Partial Fulfillment of the Requirements for the

Degree of

DOCTOR OF PHILOSOPHY

IN ANIMAL HUSBANDRY

IN
THE GRADUATE SCHOOL

OF THE

UNIVERSITY OF ILLINOIS
1921

LIBRARY OF CONGRESS |
RECEIVED

MAY 261922

' POCUMENTS Dev sn:

TABLE OF CONTENTS

I. Amino Acid Content

Vs Se Banas OVS Re he (oy gy Oe chan SI SIEVE apt ee a oe
Zeer Me COG Se Esta] Oy eC eae te
Sem DISCUBSIONTOLRCSUILS. Aut e Seater eo Os Se
4. Summary : ASIN A Nh rN ec cel ASL Re i eee
5. References -

Il. Nutritive Value

1, Review of Previous Work
Methods Employed
Discussion of Results

Summary

Apa ese)

References

Reprinted from JouRNAL or Dairy ScIENCE
Vol. IV, No. 5, September, 1921

THE PROTEINS OF COTTONSEED MEAL!

I, AMINO ACID CONTENT

W. B. NEVENS
From the Department of Animal Husbandry, University of Illinois, Urbana

I. INTRODUCTION

One of the earliest references to the proteins of cottonseed
meal was made by Ritthausen (1), who separated the pro-
teins in the form of spheroids. Osborne and Voorhees (2)
isolated a protein from cottonseed meal which had the nature of
a globulin, being soluble in salt solutions, and comprised 42.3
per cent of the total nitrogen of the meal. Another protein
(or proteins) was found to be insoluble in salt solutions but
soluble in 0.2 per cent potash solution and amounted to 44.3
per cent of the total nitrogen of the meal. Two per cent of the
total nitrogen was present in the form of water soluble proteose.

TABLE 1
Percentage of nitrogen in the different groups in various proteins

N In
N as NON- MeO

SOURCE ammo-| *3°°° | Basie |e | NS

ae TATION
Globulin, cottonseed........... ES es aE MRM EN 3 1.92] 5.71 | 11.01 18.64
Globulin sw heaGey ae yi aes tc layed eee Ge, 1.42 | 6.83 | 9.82) 0.28 | 18.39
BRIAN TOON WZ ie eA Et al PI 2.97 | 0.49 | 12.51) 0.16 | 16.13
- Hordein, barley.............. ai Seana i a TENA 4.01 | 0.77 | 12.04) 0.23 | 17.21

The distribution of nitrogen in various protein bodies was
studied exhaustively by Osborne and Harris (3), who employed
the modified Hausmann method (4). The following values are
typical of their results.

1 The results presented in this paper formed part of a thesis submitted to the

Graduate School of the University of Illinois in partial fulfillment of the require-
ments for the degree of Doctor of Philosophy in Animal Husbandry.

375

JOURNAL OF DAIRY SCIENCE, VOL. Iv, NO. 5

316 - WW. B. NEVENS

These investigators state that ‘“‘This wide variation in the
proportion of basic decomposition products of the various pro-
teins. . . . raises important questions regarding their food
value.’’ Osborne (5) further found that the basic nitrogen of
the globulin of cottonseed, as determined by precipitation with
phosphotungstic acid, consisted of 3.46 per cent histidine, 13.51
per cent arginine and 2.06 per cent lysine.

The content of the mono-amino acids of the ‘‘edestin’’ of —
cottonseed meal was determined by Abderhalden and Rostoski
(6) by the use of the Fischer ester method (7), and is as follows,
calculated for dry, ash free edestin of cottonseed:

per cent
Gly coco eee EPPERSON LOO ER Me eer ge ee a 1.2
NUE OW OEE eer ee AA een ry URC TUL hn hae em 4.5
Aminocvalerianic acid wee Sheek ee es ae aa ere Present
LAS Oy (0) U0 S AO OA a nS eR Na Raa Ina RAIA AMAT NMC LLS le 2.3
TE CU CIN ee EEN OSLER Ne DUG SE Se OU Cea ONS Re Re 15.5
Glutamiciacid eee ss NGG EEN Ne UCR: eal eae nee Aa aR 17.2
VASPATCIC ACI 2 2. le SON RTS S UES at A on a 2.9
Phenylalanine is i ciclo e eet onsale ales akc cease A 3.9
POLED ORIN LEE ASP PU UA re EI AS a RE Ae UN te 0.4
PSV TOSI MUG SORE ls URC UCI Re DUN SSR URAC IS EAR eee oe eer 2.3
AETV PCOPHANE se Were Ce eyo A LUN ee EVA ale Gc Oa Maan Present

The quantitative determination of the amino acids of feeding-
stuffs by means of the Van Slyke method (8) was undertaken
by Grindley and his co-workers (9), and a little later by Nollau
(10). In Nollau’s procedure, samples of the finely ground feeds
were hydrolyzed with 20 per cent hydrochloric acid until the
content of amino acid, as determined by the Van Slyke method,
became constant. ‘The material insoluble in hydrochloric acid
was filtered off, the clear extract concentrated under diminished
pressure and made up to a certain volume. The total nitrogen
content of this extract was used as a basis for calculating the
final results. In a report of the subsequent work of Grindley
and co-workers (11), it is claimed that since Nollau filtered off
the solid residue after hydrolysis of the feedingstuff and before
making his total nitrogen determinations upon which the final
calculations were based, that his results are not accurate since
a part of the total nitrogen was undoubtedly discarded in the
solid residue.

THE PROTEINS OF COTTONSEED MEAL BT

The heats of combustion of several vegetable proteins were
carefully determined by Benedict and Osborne (12). The
globulin of cottonseed was found to yield 5596 calories per
gram, compared to 5358 calories per gram for the globulin of
wheat and 5916 calories per gram in the case of the hordein of
barley. In commenting upon their determinations, the inves-
tigators state that ‘‘many irregularities. . . . appear, which
are doubtless due to the different proportion of the various amino
acids which constitute the molecules of the different proteins. ”’

It is evident from the foregoing discussion that our knowledge
of the composition of the proteins of cottonseed meal is very
incomplete. The globulin is the only protein of cottonseed
which has been isolated in pure form and whose composition
has been determined. The globulin, howéver, according to
Osborne and Voorhees, contains only 42.3 per cent of the total
nitrogen of the cottonseed. The character, identity and chemical
composition of the remaining proteins are practically unknown,
and it is evident from the data given above that our knowledge
of the distribution of the nitrogen in the proteins of cottonseed
meal is very meager indeed. The investigation of the distribu-
tion of the nitrogen in the proteins of cottonseed meal therefore
constituted the object of this study...

II. METHODS EMPLOYED IN CHEMICAL ANALYSIS?

The method of analysis employed consisted of two main
procedures. The first consisted of a series of extractions whereby
the nonprotein together with a very small amount of protein was
first removed, and following this, the proteins were extracted
from the residual matter of the sample which consisted mostly of
fiber. The second main procedure embraced the hydrolysis of
the extracted proteins, and the analysis of the resulting solution

2 The method of procedure here outlined is one which has been developed and
perfected in this laboratory by Dr. H. 8. Grindley, Mr. T. S. Hamilton and asso-
ciates (9, 11, 33, 36 and unpublished manuscripts). The method of extraction
preliminary to hydrolysis of the proteins has been developed entirely in this
laboratory, while the actual determination of the nitrogen in the different groups
follows closely the method of Van Slyke, but includes modifications perfected
in this laboratory.

378 WwW. B. NEVENS

for certain amino acid and other groups according to the general
method of Van Slyke (8).

The sample of cottonseed meal was prepared from good
quality commercial meal, finely ground and passed through a 40-
mesh sieve. Hach sample taken for analysis weighed 15 grams
and contained 1.0194 grams of nitrogen, or about 6 grams of
protein.

In the first three extractions, which were carried out con-
secutively, cold anhydrous ether, cold absolute alcohol and
cold 1.0 per cent trichloracetic acid were used. The samples of
feeding stuff were placed in 500 cc. centrifuge bottles and 100
to 200 ce. of the reagents added. The bottles were placed on
a shaking machine which rolled them back and forth continually.
Usually two extractions were made each twenty-four hours, one
extraction period being seven to eight hours and the other
15 to 16 hours in length. At the end of the extraction period,
the sides of the bottles were washed down with the reagent, the
bottles centrifuged and the clear supernatant liquid decanted.
Usually six or seven extractions with each reagent were necessary.

The ether and alcohol extracts were filtered and any residues
returned to the centrifuge bottles. After slight acidification
with sulphuric acid, the ether and alcohol were evaporated and
recovered and total nitrogen determinations made on the residue.
The small amount of protein removed in the trichloracetic acid
extracts was recovered by precipitation with colloidal ferric
hydroxide (containing 5 per cent Fe,O;) in boiling solution.
The precipitate was transferred to a digestion flask with 20 per
cent hydrochloric acid, and total nitrogen determined in the
filtrate.

The bulk of the proteins was removed from the residue re-
maining after extraction with 1.0 per cent trichloracetic acid
by extraction, first, with dilute sodium hydroxide solution, then
with 20 per cent hydrochloric acid followed by treatment with
5 per cent sodium hydroxide solution. The dilute sodium
hydroxide solution used during the shorter period was a 0.2 per
cent solution and that during the longer period a 0.1 per cent
solution. These extracts were neutralized, acidified with hydro-

THE PROTEINS OF COTTONSEED MEAL 379

chloric acid and concentrated in vacuo to a small volume. An
equal volume of concentrated hydrochloric acid was then added.
The residues remaining after treatment with dilute sodium
hydroxide solution were boiled for three minutes with 20 per cent
hydrochloric acid. After cooling, the solution was filtered
off, the residue washed and the procedure repeated once. The
washings were evaporated to a small volume, an equal volume
of concentrated hydrochloric acid added and the washings then
combined with the main hydrochloric acid extract. The residues
insoluble in hydrochloric acid were treated three times with 5
per cent sodium hydroxide solution using centrifuge bottles as
in former extractions. After washing the residues nearly free
from alkali, they were submitted to Kjeldahl analysis. The
extracts and washings were acidified with hydrochloric acid,
concentrated in vacuo and transferred to digestion flasks with
an equal volume of concentrated hydrochloric acid.

The proteins precipitated by colloidal iron and the proteins
removed by extraction with dilute sodium hydroxide, 20 per
cent hydrochloric acid and 5 per cent sodium hydroxide were
completely hydrolyzed by boiling for fifteen hours upon a
combined electric plate and sand bath under reflux condensers.
The resulting solutions were combined and analysis for the
chemical groups characteristic of certain amino acids executed
essentially as directed by Van Slyke (8), but with the use of
minor improvements perfected in this laboratory.

III. DISCUSSION OF RESULTS

The results obtained by application of the method of chemical
analysis outlined in the preceding section to eight portions of the
same original sample of cottonseed meal are shown in the accom-
panying tables 2 and 3. Table 2 shows the values expressed in
percentage of the total nitrogen present in the sample of feeding
stuff when it was taken for analysis, while table 3 shows the same
values expressed in percentage of the feeding stuff itself.

Two averages are included in the tables. The first is com-
piled by taking the average of all values obtained by analysis of

the entire eight samples. The second average is obtained by

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383

384 W. B. NEVENS

averaging the results secured in the analysis of the complete
samples C2, C3, C6 and C7. It is believed that the latter
average more nearly expresses the actual composition of the
commercial cottonseed meal used, for the following reasons:
(a) These samples, i.e., C2, C3, C6 and C7 show the best agreeing
results throughout. The two parts of sample C1 agree well in
the amount of arginine nitrogen, but show a considerable differ-
ence in the amounts of amino nitrogen, non-amino nitrogen and
histidine nitrogen. The totals of the nonprotein nitrogen plus
the protein nitrogen are considerably below the average of all the
samples. In sample C4 the non-amino nitrogen is particularly
low, this value being one of the principal factors contributing
to the noticeably low nonprotein plus protein of this sample.
Sample C5 is omitted from the average partly on account of the
non-agreement of its arginine nitrogen and histidine nitrogen
values. Of the latter values, one is 3 per cent above the average
of all samples. (b) ‘The second average, i.e., of samples C2,
C3, C6 and C7, includes values which are most free from obvious
errors. In making the determinations in the case of sample
C4, two determinations were lost, and in the case of sample C5,
one determination was lost. While the results obtained for sam-
ple C8 agree fairly well throughout with themselves and with the
average, the results are consistently high, and it is excluded
from this average on the grounds of the totals obtained, which
are obviously too high.

Nonprotein nitrogen. The first section of tables 2 and 3shows
the amount of nitrogen removed in the preliminary extractions
with absolute ether, absolute alcohol and trichloracetic acid.
While the absolute ether in the cold is used primarily to remove
the lipins, such as the oils, waxes, etc., it also dissolves various
amounts of other substances, such as coloring matters, and at the
same time a small amount of nitrogen. The thorough extrac-
tion with absolute alcohol following the treatment with absolute
ether presumably completes the extraction initiated by ether.
The alcohol removes somewhat more nitrogen than the extrac-
tion with ether. The bulk of the nonprotein nitrogen accounted
fer, about 89 per cent of the total, remains, however, in the tri-

THE PROTEINS OF COTTONSEED MEAL 385

chloracetic acid extracts after precipitation of the proteins by
colloidal ferric hydrate and the removal of the precipitate by
filtration. ‘That the nitrogen determined in these latter extracts
is not protein nitrogen is apparent from the work of Van Slyke,
Vinograd, Vilchur and Losee (138), Hill (14), Wolff (15),and
others.

It seems from the study of the character of the nonprotein
nitrogenous constituents of feedingstuffs by Grindley and
Kekstein (16) that the forms of nitrogen represented in this
classification consist principally of those. forms naturally re-
sulting from the cleavage of the proteins upon hydrolysis and
therefore could not interfere in the determination of the charac-
teristic chemical groups of the proteins were they not removed
in the preliminary extractions. Neidig and Snyder (17), who
recently determined the proportion of nitrogen in the form of
ammonia in the ether extracts and alcohol extracts of different
kinds of silage, found that from 28.1 per cent to 100 per cent of
the ether extract nitrogen consists of ammonia nitrogen, while
from 14.2 per cent to 23.4 per cent of the alcohol extract nitrogen
is yielded as ammonia nitrogen. This indicates that only a
part of the nitrogen soluble in ether and alcohol would appear
in the ammonia fraction were it not removed previous to hydrol-
ysis. Therefore, the removal of the nonprotein nitrogen at
this point avoids possible complications in the further prose-
cution of the analytical procedure, and it is believed that the
accuracy of the further determinations has been increased over
that of previous methods by the removal of the nonprotein
nitrogen before hydrolysis of the proteins. The total amount of
the nonprotein nitrogen present in cottonseed meal found by
the method used amounted to 6.106 per cent of the total nitrogen
contained in the feedingstuff.

Results of the Van Slyke analysis. It is a matter of common
knowledge that one of the important sources of loss in the analy-
sis of proteins by methods involving the employment of acid
hydrolysis is the formation of an insoluble black substance called
humin. The term melanin is also applied to this substance,
on account of its supposed relationship or similarity to the

386 W. B. NEVENS

naturally occurring body pigments. The amount of humin formed
in acid hydrolysis of the proteins is greatly increased by the
presence of carbohydrates, as shown by Gortner and associates
(18, 19), Hart and Sure (20), and Osborne, Van Slyke, Leaven-
worth and Vinograd (21). A part of the humin formed, how-
ever, remains in solution in the hydrochloric acid, and is
termed soluble humin. |

In these experiments the soluble humin which is adsorbed by
the lime used in neutralizing the hydrolysate when determining
ammonia nitrogen, carried with it a larger amount of nitrogen
than the insoluble humin. The sum of the insoluble plus the
soluble humin nitrogen found amounts to 6.589 per cent, which
constitutes no inconsiderable error, since at present it 1s im-
possible to determine the character of the nitrogen discarded in
this form.

It may be noted by referring to table 1 that the amount of
soluble humin nitrogen in the first four samples is considerably
greater than in the succeeding four. Possibly this is due to a
slight variation in the analytical procedure. In the case of
samples C1 to C4, inclusive, it was necessary to add 75 to 90
ec. of calcium hydroxide in order to neutralize the hydrochloric
acid before distillation of ammonia. About 20 cc. in excess were
then added. With the next four samples evaporation of the
acid hydrolysate in vacuo was continued longer in order to
drive off a greater proportion of the hydrochloric acid. In conse-
quence, only 30 to 40 cc. of calcium hydroxide were necessary
to effect neutralization, and in these cases only a small excess,
about 10 cc., of calcium hydroxide was added. The hypothesis
is put forward that the presence of a large excess of calcium
hydroxide during the distillation of ammonia may result in the
adsorption of some amino acid nitrogen which is incompletely
removed in the subsequent washing of the sticky mass.

When it is recalled that the proteins of cottonseed meal form
approximately 43 per cent of the feedingstuff, the amount of
nitrogen in the humin resulting from the hydrolysis of these
proteins, as determined in these experiments, is not excessive
when compared to the amounts obtained in the hydrolysis of

THE PROTEINS OF COTTONSEED MEAL 387

pure proteins by Van Slyke (8), some of whose results are shown
in the accompanying table.

The amount of nitrogen recovered as ammonia was quite
constant in all the samples. Little can be said in regard to the
significance of this fraction, aside from the fact that the propor-
tion of the total nitrogen of cottonseed meal which appears as
ammonia is quite in harmony with that of other feedingstuffs.
A particularly characteristic feature of the amino acid con-
tent of cottonseed meal is the remarkably high content of argi-
nine. This is much higher than that found in any other feeding
stuff so far examined, with the exception of peanuts, although it
is not so high as that found in some other vegetable proteins.
Van Slyke (8) found 27.05 per cent arginine nitrogen in edestin

TABLE 4

Amounts of humin nitrogen in pure proteins expressed in percentage of the total
nitrogen of the protein

PROTEIN AND DESCRIPTION HUMIN NITROGEN
per cent
Ghadinhfromy wheat yee wees echoes nes i ie mua 0.86
SETLES Carne yee He CH re GIES Sy Raye Our a A UN UE RY UL NR PN 1.838
Bal rimy (Merekes)/ eset crtye Ce far - aeiciovels)ie(eye a) Nioie\ alain 3.43
Oxyhemoglobin (‘‘pure, crystalized’’)..............3..... 3.60
Dog’s 113 15 peep eeOUE ODE EDO node un dd aonenoedenncd aaa 7.35

while Nollau reported that hemp seed, peanuts, black walnuts,
and hickory nuts have an arginine nitrogen content of more
than 20 per cent.

In the Van Slyke procedure the only amino acids deter-
mined by direct analysis are arginine and cystine. Just how
much importance may be attached to the results obtained for the
latter is questionable, even though the values found in the
different samples do not vary widely. These values, however,
probably fall short of the true value, due to losses in the deter-
mination of cystine. Van Slyke (8) has shown that boiling
cystine for sixteen hours with hydrochloric acid resulted in the
conversion of one-half of its nitrogen into forms not precipitable
by phosphotungstic acid. Since in the analytical procedure de-
scribed above, hydrolysis of the proteins was carried out by

388 W. B. NEVENS

boiling them with 20 per cent hydrochloric acid for fifteen hours,
it is probable that much of the cystine was destroyed during
that reaction. If it is assumed that the correct value for cys-
tine should be double that actually obtained, then the total
nitrogen of the bases would amount to 31.75 per cent of the
total nitrogen of the feedingstuff.

The values given for histidine and lysine are somewhat vari-
able among the different samples. These variations are likely
due in large measure to the indirect method used in their deter-
mination, since slight errors in any or all of the three direct
determinations of arginine nitrogen, cystine nitrogen and the
total nitrogen of the bases are doubtless all reflected at these
points.

The content of monc-amino acid nitrogen of cottonseed meal
is considerably less than that found in other feedingstufis,
possibly due to the greater proportion of the total nitrogen
which is formed by the basic amino acids. One of the interest-
ing features of the results of the Van Slyke analysis of the pro-
teins of cottonseed meal is brought out in the summation of the
ammonia nitrogen, the nitrogen of the bases, mono-amino
acid nitrogen, and non-amino acid nitrogen, the four groups
which represent the total content of strictly amino acid nitro-
gen as determined by this method. The sum of these is 83.132
per cent. While this sum is not so great as that in the case of
some other feedingstufis or of animal proteins, as determined by
previous investigators employing the Van Slyke method of
analysis, it is a much larger amount than it was possible to
secure in most cases from comparable sources by the methods of
isolation and purification employed by the earlier investigators.
Thus the tabulations of Lusk (22), combining the results of
Osborne and associates in this field, show the maximum amino
acid content of zein of maize, to be 88.87 per cent and that of
gliadin of wheat as 85.68 per cent, but in the majority of cases
the sum of the nitrogen content of the amino acids actually
isolated from vegetable proteins ranges from 50 to 65 per cent
of the total nitrogen of the protein.

THE PROTEINS OF COTTONSEED MEAL 389

Uncharacterrzed mitrogen lost in analysis. In the various
steps of the analytical procedure small amounts of nitrogen of
unknown character are included in residues and solutions which
are discarded. In general, these have been disregarded by
workers in other laboratories, especially those losses occurring
at points indicated in table 2 by the last four of the subheadings
included under the heading ‘‘Uncharacterized nitrogen lost in
analysis,’’ but in this laboratory the nitrogen discarded at each
of these steps has been determined. Under the above mentioned
headings, it is shown that, on the average, only 0.492 per cent
of the total nitrogen remains in the residues insoluble in strong
- alkali, or in other words 99.508 per cent of the total nitrogen
of the feedingstuff is extracted as a result of the method employed,
and that in individual cases as much as 99.78 per cent of the
total nitrogen present was removed. As previous workers
failed to isolate the proteins from the feedingstuff before hy-
drolysis, it is not established that part of the insoluble residue
discarded in their methods did not include some nitrogen in the
form of non-hydrolyzed protein although this does not seem
highly probable. It is believed, however, that the nearly
complete extraction of the proteins before hydrolysis lends to the
accuracy of the method by facilitating Pydrolyts and in reducing
the amount of humin.

The largest item of loss occurs in the residue which remains
after dissolving the precipitate of the bases in the amyl alcchol-
ether mixture. This, presumably, is soluble humin which has
not been adsorbed by the lime in the determination of ammonia,
and fouls the solution at this point. This difficulty was also
encountered by Menaul (23), who employed a preliminary
precipitation with phosphotungstic acid in boiling solution for
the separation of the humin and ammonia before the precipita-
tion of the bases. In the present investigation, very little of the
soluble humin appeared when the bases were precipitated, in
most cases the precipitates being free from black particles.
Washing with alternate portions of amyl alcohol-ether and
water and then taking up the residue and washing thoroughly
with water seemed to have little effect in reducing this source

390 W. B. NEVENS :

of loss. A considerable portion of the nitrogen lost is soluble
in the amyl alcohol-ether mixture, while smaller losses occur
in the residues resulting from concentration of the solutions of
the bases and filtered from the bases. Presumably, the second,
third and fourth items of loss include some nitrogen which
should be credited to the bases, but the character of this nitrogen
was not determined. If these losses can be reduced, the total
nitrogen of the bases of cottonseed meal may be found to be
somewhat greater than the amount here reported.

' Total nitrogen accounted for. Summation of the nitrogen
found in the various fractions of the protein molecule together
with that in the unavoidable losses in the procedure gives totals
which average 98.75 per cent. While the use of the Van Slyke
method of analysis has enabled others to account for as great
a proportion of the nitrogen of feedingstuffs, it is doubtful,
for reasons pointed out below, if their results give as accurate a
picture of the distribution of nitrogen in feedingstufis as is
obtained by the procedure employed in the present investiga-
tion.

Physiological significance of the basic amino acids. Our knowl-
edge of the physiological réle of arginine and histidine has been
enhanced by the studies of Ackroyd and Hopkins (24). Em-
ploying rations in which the nitrogen was provided in the form ~
of hydrolyzed casein from which these two amino acids had
been removed by precipitation according to the method of
Kossel and Kutcher, it was found that rats receiving these
rations declined rapidly in weight, but that when either amino
acid was returned to the ration, loss in weight was prevented and
some growth ensued. The investigators suggest that possibly
either of these amino acids may be converted into the other
by the animal body. It was further observed that when argi-
nine and histidine are removed from the ration, the excretion of
allantoin, which is the main end product of purine metabolism
in the rat, was lowered. Subsequent experiments proved that
the falling off of allantoin excretion was not due to lowered me-
tabolism. From these observations and from the fact that the
arginine, histidine and guanine molecules have similar structural

THE PROTEINS OF COTTONSEED MEAL 391

relationships, it was concluded that possibly one of the functions of
arginine and histidine is to furnish the raw material for the purine
metabolism of the animal organism.

The above conclusion regarding the importance of arginine in
purine metabolism is given added weight by the findings of
Myer and Fine (25) regarding the creatine content of muscle.
Differences of as much as 2.5 per cent in the creatine content of
muscle were noted as a result of feeding rations high and low in
arginine.

That cystine plays an important part.in nutrition has been
brought out by several investigators, among them Osborne and
Mendel (26). The latter obtained adequate growth by the
addition of cystine to rations containing 9 per cent of casein, on
which growth had been limited. Geiling (27), working in this
laboratory, concluded that cystine seems to be necessary for the
maintenance of adult mice. ‘The importance of cystine to the
animal organism is admirably set forth by Matthews (28).

In the intermediary metabolism of the body, that is, the metabolism
of the tissue, sulphur probably plays a very important réle. This is
shown not only by the fact that it is absolutely necessary for the
continued existence of the body, as necessary as nitrogen or any of the
other elements, but also by the fact that it is one of the most labile
elements of the protein molecule. No other element is split off from
the proteins with greater ease than this. It is, indeed, the labile
element par excellence. Moreover, cysteine, which is one of the amino
acids, readily oxidizes itself. It is a reducing body. It oxidizes
spontaneously and there are many points in its oxidation which strongly
resemble the process of respiration. Thus the most favorable concen-
tration of hydrogen ions for the oxidation of cysteine is the same as
that in protoplasm; both cysteine and protoplasm are poisoned by
many of the same substances, such as the nitriles, the cyanides, acids,
and the heavy metals; their oxidations are catalyzed or hastened in
the same manner by iron, arsenic and some other agents. For these
reasons it has been suggested by Hefter and the author that there is
more than a superficial connection between the oxidation of cysteine
and the respiration of the cell.

392 W. B. NEVENS

The necessity of lysine for growth has been conclusively dem-
onstrated by Osborne and Mendel (29). When gliadin of
wheat, which contains only a minute amount of lysine, formed
the sole source of protein in the rations of rats, the live weight of
the animals was maintained over long periods, but normal
erowth could not be secured. When lysine was added to the
rations, normal growth occurred. In other investigations (80),
in which zein of maize was used as the source of the protein,
it was found that a rat could be maintained at an almost constant
weight of 50 grams for a period of one hundred and eighty-two
days when tryptophane was added to the extent of 3 per cent
of the zein. The further addition of lysine induced normal
growth. Further study (31) of the necessity of lysine in the
ration convinced these investigators that about 2 per cent of
the protein of the ration must consist of lysine in order to pro-
mote normal growth in the rat. Osborne and Mendel (82) also
demonstrated the necessity of lysine for the growth of chickens.

In view of the essential rdle which the basic amino acids play
in nutrition as brought out above, it is reasonable to assume from
a survey of the analytical results of cottonseed meal secured in
this investigation that the proteins of this feedingstuff have a
high nutritive value. The combined arginine and histidine
content of cottonseed meal is greater than that of any other
feedingstuff so far analyzed with the exception of the peanut.
This feature alone is of great importance in view of the fact
that arginine and histidine seem to be interchangeable in nutri-
tion. While the lysine content cannot be said to be exceptional
in any particular it seems apparent from the above discussion,
that the combined proteins of cottonseed meal contain sufficient
amounts of both cystine and lysine to render them adequate for
nutrition.

Comparison with previous analyses of cottonseed meal. As
shown in the introduction, there are but few determinations of
the chemical composition of the proteins of cottonseed meal.
The earliest studies were made upon one of the isolated pro-
teins, the globulin or ‘‘edestin’”’ of cottonseed, the results of
which can not well be compared to analyses of the combined

THE PROTEINS OF COTTONSEED MEAL 393

proteins, since, as previously mentioned, the globulin contains
but 42.3 per cent of the total nitrogen of cottonseed meal. In
the accompanying table 5, the values secured by two previous
investigators who made analyses of the combined proteins of
cottonseed meal are brought together for comparison with those
obtained in this investigation.

It is evident from the results presented that there is a general
agreement between the three sets of values, but that there are
considerable differences in several important particulars. As
pointed out in the introduction, Nollau (10) calculated his re-
sults upon the total nitrogen content of the hydrolyzed solution
after filtering off the solid residue insoluble in hydrochloric

TABLE 5
Distribution of nitrogen in cottonseed meal as determined by different investigators
(Results expressed in percentage of the total nitrogen of the feeding stuff)

amino | NON-

nee iene TOTAL
INVESTIGATOR aa oN NEN) (Cueto ee BNE Teens a as ee Naa
FROM | Trou | ED FOR

BASES |) aaa
Nol au ee 6.27 | 14.06) 12.77) 2.74 | 7.57 | 1.94) 45.02) 7.49 | 97.48
Crindleye sees eee 7.78 | 10.45] 19.52] 0.65 | 5.47 | 4.78! 42.82] 5.43 | 96.90

INEVENSicytesier Ns): 6.58 | 9.49) 18.74) 0.91 | 7.40 | 3.81) 40.12) 2.68 | 98.751

1 Includes 9.03 per cent N removed in preliminary extractions plus unchar-
acterized nitrogen lost in method of analysis.

acid. This means that all of his calculations are too high,
since a part of the nitrogen of the sample was undoubtedly dis-
carded in the solid residue. The value of 6.27 per cent humin
nitrogen reported by Nollau must, therefore, represent the
soluble humin nitrogen, which is nearly as large a value as
that obtained by the writer for the sum of the insoluble humin
nitrogen plus the soluble humin nitrogen. The amount of
soluble humin nitrogen found by the writer was but 3.89 per
cent. Compared to the total humin nitrogen found by Grindley,
et al. (9), the amount of total humin nitrogen as determined
by the writer was 1.19 per cent less. The reduction of the
humin nitrogen has no doubt been an important contributing

394 WwW. B. NEVENS

factor in the present investigation in securing somewhat higher
values of the basic amino aids. In view of the known effects
of acid hydrolysis of the proteins in the presence of carbohy-
drates, as already pointed out, it is reasonable to assume that
the smaller amount of nitrogen discarded in the form of humin
in these experiments than in those of Grindley may be attributed
to the more complete separation of the proteins from the carbo-
hydrates before hydrolysis.

The method of analysis of the proteins after hydrolysis by
hydrochloric acid, as employed by Grindley et al., was similar
to that employed by the writer, the main point of difference
between the complete procedures being in the omission by the ©
former workers of the extractions previous to hydrolysis. At
just what point the 6.106 per cent of nonprotein nitrogen re-
moved by the writer in the preliminary extractions might
appear were it not so removed, is not clear. However, the sum
of the ammonia nitrogen, amino nitrogen and non-amino nitro-
gen in the filtrate from the bases, obtained by Grindley et al, is
6.414 per cent greater than the sum of the corresponding values
obtained by the writer, so it is possible that these three forms of
nitrogen as reported by the former comprise some nitrogen not
derived from the proteins as such.

It is evident from the table that the nitrogen of the bases as
found by Grindley and his coworkers are in much closer agree-
ment with those obtained by the writer than those reported by
Nollau. The latter’s figures for arginine are obviously too
low, while his cystine values are more than four times as great
as those of Grindley et al. and three times as great as those of
the writer. Accordingly, the lysine nitrogen values as calculated
by Nollau are correspondingly too low. The values for the total
nitrogen of the bases as found by the three investigators in the
order given in the table are as follows: 25.02 per cent, 30.42
per cent and 30.84 per cent respectively, the last being nearly
0.5 per cent higher than previous determinations.

The total nitrogen accounted for in the three reports is like-
wise shown to be 97.86 per cent, 96.90 per cent and 98.75 per cent.
The greater amount in the last case is evidently due in part

THE PROTEINS OF COTTONSEED MEAL 395

at least, to the inclusion of the determinations of the uncharac-
terized nitrogen lost at points were unavoidable losses occur in
the method of analysis. ‘These losses were not determined by
the first two investigators.

Comparison of the distribution of nitrogen in cottonseed meal
with that in other feedingstuffs. A comparison of the results of
analysis of the proteins of cottonseed meal, as discussed above,
with those obtained by Hamilton, Grindley and Nevens (33)
for alfalfa hay, oats and corn is of value in studying the relative
nutritive value of the proteins of these feedingstuffs, as well as
the applicability of the general method of analysis to feeding-
stuffs which vary widely in composition. In the analysis of
oats and corn an additional preliminary extraction, which
involves the use of hot trichloracetic acid, is employed to remove
the starch. This extraction is not necessary in the case of
cottonseed meal and alfalfa hay on account of the absence of
starch in the former, as stated by Withers and Fraps (84),
and the relatively small amount of starch in the latter.

The first point of interest in contrasting these feedingstuffs, as
may be noted by reference to table 6, is their content of non-
protein nitrogen.- Oats contain more than twice as much non-
protein nitrogen as cottonseed meal, while alfalfa hay contains
more than three times as much. Hart and Bentley (35) found
that 23.5 per cent of the nitrogen of alfalfa hay is present in a
water soluble form, while Grindley and Eckstein (16) found a
value of 28.4 per cent for the same feedingstuff.

The amount of total humin is greatest in the case of alfalfa,
a natural result, since the proteins are more difficultly extracted
from those feedingstuffs containing large amounts of crude fiber.
The amount of humin in the case of corn is very small indeed,
considering the high percentage of carbohydrates in this cereal,
and compares very favorably with the amounts of humin re-
sulting from the hydrolysis of pure proteins as shown in table 3.
Cottonseed meal occupies a medium position in respect to the
proportion of humin nitrogen.

The most striking difference between these four feeding-
stuffs is in their basic amino nitrogen content. Cottonseed

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THE PROTEINS OF COTTONSEED MEAL 397

meal, as already indicated, is exceptionally high in arginine nitro-

gen, but it is also much higher in its total basic nitrogen con-
tent than the other three feedingstuffs, the values for the four
feedingstuffs being: alfalfa hay, 17.412 per cent; oats, 21.228
per cent; corn, 17.529 per cent; and cottonseed meal, 30.846 per
cent. The sum of the arginine nitrogen and histidine nitrogen is
more than twice as great in the case of cottonseed meal as in
the case of alfalfa hay and nearly twice as great as that of corn.
From the considerations presented above regarding the biological
significance of the basic amino acids, it would be logical to
assume that these wide differences in the chemical composition
of the proteins of different feedingstuffs indicate similar differ-
ences in their nutritive value, though probably not in corre-
sponding degree. This point is mentioned in another paper in
connection with the discussion of the results of the feeding
experiment conducted for the purpose of studying the nutri-
tive value of the proteins of cottonseed meal.

Alfalfa hay contains the smallest proportion of mono-amino
acid nitrogen, possibly owing to its high content of nonprotein
nitrogen while corn is exceptionally high in its content of
both mono-amino and non-arhino acid nitrogen.

The largest amount of nitrogen lost in the method of analy-
sis occurs in the case oi alfalfa, which is accounted for largely
in the nitrogen remaining in the residues after the preliminary
extractions have been completed. The next largest amount
is in the case of corn, where the bulk of the loss is due to un-
adsorbed humin. The nitrogen is extracted very completely
from both oats and corn. Cottonseed meal occupies a medium
position with respect to the nitrogen lost in the analytical
procedure.

The total nitrogen accounted for in the case of the various
feedingstufis is a point worthy of special note. The total is
least in the case of alfalfa and greatest with corn. Here again
cottonseed meal occupies a medium position. In this rather long
method of analysis, which involves many extractions, concentra-
tions, precipitations, filtrations and transfers, and which at some
stages renders the proteins subject to putrefaction unless care

398 W. B. NEVENS

is taken, only 0.101 per cent of the total nitrogen originally
present in the sample of corn was not accounted for, a very
remarkable result indeed.

An examination of the results of individual analyses of the
four samples of alfalfa hay and six samples each of oats and corn
which were averaged to obtain the values shown in table 6,
brings out the fact that the analytical results in the case of each
of these feedingstuffs show, on the whole, less variability than
the values for the eight samples of cottonseed meal shown in
table 2. At least two factors operated to effect the difference.
The analyses of the first three feedingstuffs mentioned were
conducted by persons experienced in the manipulation and
execution of the Van Slyke analysis and the analyses used for
the averages were selected from a number of analyses. The
analyses of cottonseed meal were made by the writer who had
had no previous experience in the conduct of the Van Slyke
method, and the analyses presented in table 1 are the entire
results of the work. These considerations are strong evi-
dence that the method of analysis here described is of general
application to feedingstuffs and may readily be carried out.

Summary of the discussion of the results of the chemical analysis
of the proteins. ‘The accuracy of the determination of the amino
acid content of the proteins of cottonseed meal has been in-
ereased over that of previous methods by the removal of the
nonprotein nitrogen before proceeding with the hydrolysis of the
proteins.

The accuracy of the determination has been still further
‘Increased by the reduction of the humin substances formed as
a result of the hydrolysis of the proteins.

The amount of arginine nitrogen is much higher than that
in most other feedingstuffs. The sum of the four basic amino
acids is about 0.5 per cent higher than values previously found
for cottonseed meal.

The method of extraction employed was found to result in
the removal of 99.5 per cent of the total nitrogen present in the
feedingstuff.

THE PROTEINS OF COTTONSEED MEAL 399

The sum of the ammonia nitrogen and amino acid nitrogen
fractions is 83.132 per cent of the total nitrogen, an amount
comparable to the sum of the same fractions previously ob-
tained from pure vegetable proteins.

Of the total nitrogen originally present in the ample of cotton-
seed meal, 98.75 per cent was accounted for by summation of
the fractions obtained at different stages in the method of
analysis, a proportion greater than any previously reported for
the same feedingstuff.

The complete method of analysis outlined in this paper is
believed to be of general application to feedingstuffs and may
readily be executed with successful results.

The writer is greatly indebted to Dr. H. S. Grindley for many
courtesies extended during the course of this investigation in
addition to his constructive criticism and general supervision
of the work. His thanks are also due Mr. T. S. Hamilton for
advice regarding certain analytical procedures.

REFERENCES

(1) Rirruausen, H.: Jour. f. prak. Chem. (Neue Folge), 1881, xxiii, 481.

(2) Ospornez, T. B., anD VoorHEsEs, C. G.: Jour. Amer. Chem. Soc., 1894, xvi,

778; Annual Rept. Conn. Agr. Exp. Sta., 1898, xvii, 211.

(3) Ossporne, T. B., anp Harris, I. F.: Jour. Amer. Chem. Soc., 1903, xxv, 323.

(4) Hausmann, W.: Zeit. physiol. Chem., 1899, xxvii, 95; ibid., 1900, xxix, 136.

(5) Osporne, T. B.: The Vegetable Proteins. 1912, p. 59.

(6) ABDERHALDEN, E., AND RosToskI, O.: Zeit. physiol. Chem., 1905, xliv, 265.

(7) Fiscoer, E.: Zeit. physiol. Chem., 1901, xxiii, 151.

(8) Van Stryke, D. D.: Jour. Biol Chem., ix, 185; ibid., 1911, x, 15; ibid., 1912,
Xll, 275; ibid., 1915, xxii, 281.

(9) Grinptry, H. S., JosepaH, W. E., anp Suater, M. E.: Jour. Amer. Chem.
Soc., 1915, xxxvii, 1778; Sc., 1915, xlii, 70.

(10) Notuav, E. H.: Jour. Biol. Chem., 1915, xxi, 611.

(11) Grinpury, H. S., ann Suater, M. E.: Jour. Amer. Chem. Soc., 1915,
XXXVil, 2762; Proc. Soc. An. Prod., 1917, p. 133.

(12) Brenepict, F. G., anp Ospornz, T. B.: Jour. Biol. Chem., 1907, iii, 119.

(18) Van Styxz, D. D., Vinocrap-ViticHur, M., anp Losrz, J. R.: Jour.
B ol. Chem., 1915, xxiii, 377.

(14) Hity, R. L.: Jour. Biol. Chem., 1915, xx, 175.:

(15) Wotrr, C. G. L.: Jour. Physiol., 1915, xlix, 89.

400 W. B. NEVENS

(16) Grinpuey, H. S., anp Eckstein, H. C.: Jour. Amer. Chem. Soe., 1916,
XXXVill, 1425.

(17) Nerie, R. E., anp Snyper, R. §.: Jour. Amer. Chem. Soc., 1921, xliii, 951.

(18) Gortner, R. A., anp Buisu, M. J.: Jour. Amer. Chem. Soc., 1915, xxxvii,
1630; Jour. Biol. Chem., 1916, xxvi, 177.

(19) Gortnrr, R. A., anD Hoim, G. E.: Jour. Amer. Chem. Soc., 1917, xxxix,
2477 and 2736.

(20) Hart, E. B., anp Sure, B.: Jour. Biol. Chem., 1916, xxviii, 241.

(21) Osporne, T. B., Van SutyKxe, D. D., Tucano He C. S., AND VINOGRAD,
M.: Jour. Biol Chem., 1915, eT 259.

(22) Lusx, G.: The Science of N utrition. 3d Ed. 1919, p. 77.

(23) Mrnauvt, P.: Jour. Biol. Chem., 1921, xlvi, 351.

(24) Acxroyp, H., anp Hopkins, F. G.: Biochem. Jour., 1916, x, 551.

(25) Myrrs, V. C., anp Fing, M. §.: Jour. Biol. Chem., 1915, xxi, 389.

(26) OsporneE, T. B., anp Menpet, L. B.: Jour. Biol. Chem., 1915, xx, 373.

(27) Gritine, E. M. K.: Jour. Biol. Chem., 1917, xxxi, 173.

(28) Matuews, A. P.: Physiological Chemistry. First Edition, p. 813.

(29) OsBorne, T. B., anp MrenpEL, L. B.: Jour. Biol. Chem., 1914, xvii, 325.

(30) Osporne, T. B., AnD Mrenpzt, L. B.: Jour. Biol. Chem., 1915, xx, 351.

(31) Osporne, T. B., AnD Mrenpet, L. B.: Jour. Biol. Chem., 1916, xxv, 1.

(32) OsBorneE, T. B., AND MENDEL, L. B.: Jour. Biol. Chem., 1916, xxvi, 2938.

(83) Hamitton, T. S., Grinptey, H. S., anp Nevens, W. B.: Unpublished
manuscript.

(34) Wituers, W. A., AND Fraps, G. S.: Bul. 179, 1901, N. Car. Agr. Exp. Sta.

(35) Hart, E. B., anp Bentitry, W. H.: Jour. Biol. Chem., 1915, xxii, 477.

(36) GrinpLEey, H. 8., anp Eckstein, H. C.: Jour. Biol. Chem., 1919, xxxvii,
373; Science, 1915, xlii, 70.

Reprinted from JouRNAL OF Datry ScIENCE
Vol. IV, No. 6, November, 1921

THE PROTEINS OF COTTONSEED MEAL!
Il. NUTRITIVE VALUE
W. B. NEVENS

Department of Animal Husbandry, University of Illinois, Urbana, Illinois

I. REVIEW OF THE PREVIOUS WORK ON THE NUTRITIVE VALUE
OF THE PROTEINS OF COTTONSEED MEAL

Cottonseed meal and flour were found by Richardson and
Green (1) to be satisfactory sources of protein for the growth
of albino rats when these feeds furnished 18 per cent or more
protein to the ration. Mendel (2) states that normal growth
has been secured for considerable periods when the globulin of
cottonseed was fed in suitable concentration, such concentra-
tion having been determined by Osborne and Mendel (38) as
18 per cent of the ration. The latter investigators (4) found
that ‘‘Cottonseed flour forms a suitable adjuvant for the pro-
teins of corn gluten,” producing ‘‘satisfactory increments of
growth” in chickens. In further studies of the value of certain
proteins as supplements to corn gluten, these authors (5) demon-
strated that the proteins extracted from cottonseed flour by
sodium hydroxide solution were efficient supplements to the
proteins of corn gluten for the growth of rats. The use of either
the cottonseed globulin or the proteins precipitated from alkali
extracts of cottonseed flour in an amount equal to 9 per cent of
the ration resulted in ‘‘satisfactory growth’’ and when used to
the extent of 6 per cent of the ration ‘“‘considerable growth”’
was secured. ‘This is interpreted as attesting the excellent
quality of cottonseed proteins. McCollum and Simmonds (6)
report the maintenance of body weights by rats fed a ration
containing 6 per cent of protein derived from cottonseed.

1 The results presented in this paper formed part of a thesis submitted to the

Graduate School of the University of Illinois in partial fulfillment of the require-
ments for the degree of Doctor of Philosophy in Animal Husbandry.

552

THE PROTEINS OF COTTONSEED MEAL 553

In studies of the relation of the quality of proteins to milk
production, Hart and Humphrey (7) found an equality in effi-
ciency of the proteins of gluten feed, oil meal, distillers’ grains
and cottonseed meal as supplements to the proteins of corn
meal and alfalfa hay. In later experiments (8) of the same
nature, cottonseed meal proteins proved less efficient than the
proteins of gluten feed, oil meal and distillers’ grains. In these
experiments, however, the proteins of the feedingstuff tested
formed but 40 per cent or less of the protein content of the ration,
and the results were calculated upon the basis of the total nitro-
gen absorbed by the animals.

The digestibility of the proteins of cottonseed meal is stated
by Fraps (9) to be 88.4 per cent in the case of steers and sheep;
by Henry and Morrison (10) as 84 per cent, for choice and prime
cottonseed meal; by Mendel and Fine (11), who employed dogs
as experimental animals, as 67 to 75 per cent compared to 88 to
93 per cent for the proteins of meat; by Rather (12), using men
as subjects, as 77.6 per cent in contrast to 96.6 per cent for the
proteins of meat; and by Pomaski (13), who employed the gastric
juice of the dog, as 99 to 100 per cent.

From a review of the literature, it is apparent that investi-
gations upon the nutritive value of the proteins of cottonseed
meal are quite limited in extent. In the majority of experiments
cited, the investigators drew their conclusions from the main-
tenance of live weight, increase in live weight, state of health or
combinations of these criteria. In most cases the amount of
feed consumed is not recorded, so that it is impossible to judge
whether or not the results secured were due to a failure of the
animals to consume a sufficient amount of feed to cover their
energy requirements. In but one series of experiments (7, 8),°
were the conclusions based upon metabolism studies. Hence,
the further study of the nutritive value of the proteins of cot-
tonseed meal constituted the object of the present investigation.

The toxicity of cottonseed meal

Before proceeding with the investigation, it was considered
advisable to determine, so far as possible, whether or not the

554 W. B. NEVENS

toxic principle of cottonseed meal is associated with its proteins,
and further, whether cottonseed meal would prove injurious
to rats as has been found (14) in the case of many other species
of animals.

From an examination of the literature, it would seem that
there is but little basis for attributing the toxicity of cottonseed
meal to its proteins. The assumption that the high protein
content of cottonseed meal is responsible for its harmful effects
(15) was denied by Dinwiddie (16), who maintains that this
theory is not supported by a study of the recorded feeding tests.
Withers and Brewster (17) attributed the toxic principle of cot-
tonseed meal to a certain group of the protein molecule which
contains loosely bound sulphur, but later work by Withers and
associates (18) led them to conclude that the toxicity is due to
the presence of ‘‘gossypol,’”’ a definite chemical compound soluble
in ether and aniline. They believe ‘‘gossypol’’ may be changed to
a nearly related substance ‘‘D-gossypol,”’ the latter being in-
soluble in ether but soluble in aniline. When in alcoholic solu-
tion either of these compounds forms precipitates with the alco-
hol soluble proteins of wheat flour and of cottonseedmeal. They
reason that the reduction of the toxicity of cottonseed meal by
heating may be due to the inability of the animal to digest the
““gossypol”’ and ‘‘D-gossypol”’ protein compounds. The theory
that gossypol is responsible for ‘‘cottonseed meal injury”’ is
strengthened by the work of Alsberg and Schwartz (19).

In their series of feeding experiments with albino rats, Rich-
ardson and Green (1) and Osborne and Mendel (5) observed no
toxic effects, but the cottonseed kernels themselves proved
toxic.

In the light of the foregoing discussion, it seems very doubtful
if the toxicity of cotton seed meal may be attributed to either
its high protein content or to the character of the proteins which
it contains. Further, it seems clear that commercial cottonseed
meal of good quality may provide practically the entire nitrog-
enous components of the ration for albino rats over a con-
siderable period of time with no injurious effects becoming
manifest.

THE PROTEINS OF COTTONSEED MEAL 595

II, METHODS EMPLOYED IN STUDYING THE NUTRITIVE VALUE
OF THE PROTEINS OF COTTONSEED MEAL

Object of feeding experiment. The object of this phase of the
experiment was to study the nutritive value of the proteins of
cottonseed meal and to compare their nutritive value with that
of the proteins of corn and alfalfa hay for the growth of young
albino rats. It was planned to feed rations containing a medium
amount of protein, derived from the above mentioned sources,
and by means of metabolism studies to determine the extent
to which the proteins are utilized for maintenance and growth.

General plan of experiment. Young male albino rats in vig-
orous, healthy condition and having an initial weight of from
100 to 140 grams were employed. ‘The metabolism periods were
each seven days in length, two such periods following each other
without intermission with each of the experimental rations
tested. Before the first metabolism period and whenever the
rations were changed, a three day preliminary or transition period,
during which the ration to be employed during the metabolism
period was fed, was inserted. It was planned to feed the ani-
mals as large amounts of the rations as they would consume,
the daily feed allotment being slightly greater than the amount
consumed.

The rats were placed in individual glass crystallizing dishes
7z inches in diameter and 32 inches in depth, inside measure-
ments. The dishes were provided with weighted wire covers
to which were attached large test tubes fitted with rubber stoppers
and bent glass tubing, the latter extending downward through
the wire cover. The test tubes were kept supplied with ammonia-
free water. Large porcelain crucibles for receiving the feed
were supported from the covers by means of wire frames. Crys-
tallizing dishes of 60 mm. diameter were employed instead of
the crucibles for rations containing alfalfa, which were very bulky.
Ventilation was provided by means of a system of rubber tubes
which conducted a current of compressed air to the bottom of
each dish. From two to three sheets of filter paper, cut to fit
the dishes, were placed in the bottom of each dish daily to
absorb the urine.

THE JOURNAL OF DAIRY SCIENCE, VOL. IV, NO. 6

556 W. B. NEVENS

' Feces and urine were collected daily. In most cases the filter
paper absorbed the urine completely, so that the feces were nearly
always found dry. In a very few cases, particularly with ra-
tions containing alfalfa which resulted in the production of
very bulky feces, there was evidently absorption of urine by the
feces, so that it was necessary to extract the feces once or twice
with hot acidified water before collecting them. - The feces were
preserved under 95 per cent alcohol acidified slightly with sul-
furic acid. At the end of each seven-day metabolism period, the
feces were transferred to large Kjeldahl flasks and digested
according to the to the Kjeldahl-Gunning-Arnold method with
sulfuric acid, sodium sulfate and mercury. ‘The resulting solu-
tions were transferred to 500 ce. volumetric flasks and aliquots
taken for distillation.

After collecting the feces, the urine was extracted from the
filter papers by washing with a stream of ammonia-free water
acidified with sulfuric acid and held at nearly boiling tempera-
ture. The filter paper was thoroughly pulped and pressed
out after each extraction by means of a glass rod. From four
to six extractions were made, using 40 to 60 cc. of water each
time, the sides and bottom of the dish also being thoroughly
washed. The extracts were filtered through glass wool into
250 ce. volumetric flasks. The flasks were allowed to remain in
the ice box over night. The solutions were then made up to
volume at ice box temperature and transferred to 2.5 liter bottles
which were kept in a cold storage room at a temperature of
5° to 10° C. until analyzed. About 0.5 gram of powdered thymol
was employed as a preservative in each bottle in which the week’s
urine was collected. ‘The composites were thoroughly mixed and
aliquots measured out in the cold for total nitrogen determi-
nations.

The feed was weighed daily into the crucibles and mixed with a
little nitrogen-free water to the consistency of a thick paste.
The following day the feed residues were scraped out and dried
in the same oven and at the same temperature as the rations
used. In some cases the animals scattered the feed from the
crucibles about the metabolism dish. In such cases the eed

THE PROTEINS OF COTTONSEED MEAL 557

remaining in the metabolism dish at the time of collecting the
excreta was carefully separated and added to the feed residues.
When thoroughly dry the weight of the feed residues was de-
termined and the amount of feed actually consumed during the
seven-day period calculated. By previous tests in this labora-
tory it was found that the error involved in this calculation due to
a difference in the moisture content of the residues and ration
was less than I per cent, and further that the nitrogen contents
of the residues and ration were identical (20).

Preparation of rations. In preparing the experimental rations,
the starch used was first dextrinized by heating on the steam
bath after the addition of cold water and a few crystals of citric
acid. When ground corn formed one of the constituents of a
ration, it was mixed with the starch and the starch of the mix-
ture dextrinized. The other ingredients were then added, the
agar being dissolved in boiling water and added at the boiling
temperature. When necessary more hot water was added and
the ingredients thoroughly mixed. The rations were dried on
glass plates, placed above the steam bath, finely ground and
dried in an oven at a temperature of about 40°C. After drying
for several days, the rations were mixed, sampled for analysis
and placed in tightly covered glass jars.

The nitrogen free ration consisted of the following:

per cent
SHUTS w es SANE CRG SMU AO IC IN IAN 5 Se MMU EE RN ROC U LD be 5
EAU GSE rEg at ee aor eae Oana Dnt a 2 aetan 6§ 8 ah a0
SSC FOSC pees isgertel ca Were aaa U rs Re US SARIN ORV TT AGRI A Mepis 8
SARC ee mein NRC atceUMIR t om cr CUI ei ui 1 74
NORRIE SS ASA RE CaN ee kA Va AT AT 3

Water soluble vitamin, 150 mgm. of solids per 100 grams of ration.

The composition of the other rations is shown in table1. The
salt mixture used was compounded according to the formula of
Osborne and Mendel (21), while the water soluble vitamin
consisted of Osborne and Wakeman’s (22) fraction II of the
concentrated extract of the water soluble vitamin of brewers’
yeast. The stock supply of the latter was prepared in the form
of a water solution which was preserved by means of a small
quantity of chloroform and kept in the ice box. The butter-

558 W. B. NEVENS

fat was obtained by placing fresh creamery butter in large
beakers, heating to a temperature of 50° to 60° on the steam
bath, centrifuging for an hour or until the fat became water
clear, and then siphoning off the clear fat.

It was planned that all rations containing a protein feeding-
stuff should carry 10 per cent of protein (N xX 6.25), but the
actual content of protein was slightly higher, ranging from 10.38
per cent to 11.28 per cent, due to the fact that some of the con-
stituents used in making up the ration had a slightly higher
moisture content than the dried rations.

TABLE 1

Composition of experimental rations (expressed in percentage)

RATION
CONSTITUENT —————————————— eee eee

1* 2 3 4* 5 6 7

Cottonseed meal.......... 23.9 13.1 10.3 all
Cornet we em (40 32.3 | 28.4 ‘1 19.3
Allfaliaphiay yan tenes lee 63.5 40.6 | 85.7 | 29.3
Stanchiy sau ashe a ool Gee} US) NW eee = 3.0) |) ata) |) 5) .7
ALO: THEN UP CN aa 3.0 3.0 3.0
Sucrosesie sosa eee en 5.0 5.0 5:0 5.0 5.0 5.0 5.0
Buttertate: 205 von ais 10.0 | 10.0 | 10.0 | 10.0 | 10.0 | 10.0 | 10.0
Dall tsi ea ecu een imation aa 3.0 3.0 3.0 3.0 3.0 3.0 3.0

Total.................../100.0 |100.0 {100.0 {100.0 {100.0 |100.0 |100.0

Total nitrogen content.| 1.750} 1.660; 1.806) 1.777) 1.708) 1.790) 1.782

* Water soluble vitamin preparation added at the rate of 1.5 mgm. of solids
per gram of ration to rations 1 and 4.

In preparing the rations in which two or more feedingstuffs
were combined an effort was made to have each feedingstuft
furnish an equal amount of digestible protein, using the coeffi-
cients of digestibility secured in period 2 as a basis for calcula-
tion, but keeping the total content of crude protein the same
throughout the experiment, namely, 10 per cent.

The cottonseed meal used in the rations was a part of the same
sample which was employed in the analytical study presented
In a preceding paper. Through the courtesy of the Plant Breed-
ing Division of the Agronomy Department of this university a

THE PROTEINS OF COTTONSEED MEAL 009
quantity of ‘‘high protein’”’ corn containing 2.2 per cent nitro-
gen was secured, which made it possible to formulate a suitable
corn ration containing 10 per cent protein. All of the feeding-
stuffs used were in a finely ground condition before compounding
the rations.

Accuracy of metabolism work with small animals. Since the
accuracy of metabolism work depends in large measure upon the
accuracy of the collection of the excreta, especially when such
small amounts of nitrogen are involved as in the case of the
smaller laboratory animals, several experiments were carried
out to test the accuracy of the methods employed.

Each day during period 6, when 6 rats were receiving the same
feed mixture, the paper residues remaining after extraction of
the urine were collected in glass jars and placed in the ice box.
At the end of the metabolism period, the entire mass of residues,
including the glass wool used in filtration, was transferred to a
2 liter beaker and boiled for some time with about 1 liter of
water acidified with sulphuric acid. The extracts were decanted
and the procedure repeated, the acidified water being pressed
out from the residues. The extracts were filtered through glass
wool, evaporated on the steam bath and transferred to Kjeldahl
flasks for total nitrogen determination. The paper residues
also, together with the glass wool, were transferred to large
Kjeldahl flasks, and total nitrogen determined. The results of
these determinations are shown in table 2. The nitrogen ex-
tracted in the procedure described just above is assumed to be of
urinary origin and is compared to the total amount of urinary
nitrogen excreted during the week. It is shown that, as an
average of 6 such determinations, the error in the collection of
urine amounted to 2.0 per cent of the total nitrogen.

The nitrogen remaining in the paper residues which was not
extracted by boiling with acidified water was assumed to be
fecal nitrogen. In collecting the feces it was sometimes impos-
sible to entirely remove the fecal matter from the filter papers,
especially when the rations tended to cause a laxative condition.
Such a condition was not a constant effect with any ration em-
ployed, but was more frequent with rations containing alfalfa.

560 W. B. NEVENS

With the latter rations, three filter papers were generally placed
in each dish daily, while with the other rations two papers were
used. Analysis of the filter paper showed that seven filter papers
of the size used contained 1.06 mgm. of nitrogen. In making
the calculations shown in table 2 it was assumed that the nitro-
gen originally contained in the filter papers was insoluble in the
hot dilute acid employed in extraction and this has been de-
ducted from the non-extractable nitrogen remaining in the
paper residues. To the extent that such nitrogen is soluble in

: “TABLE 2
Test of the completeness of extraction of nitrogen from filter papers used as
absorbents during one metabolism period

A

1 1 [pit
he Boia eel eale ee ere (ae
Ay 2 by ga Mp 2 a A, 2 fl Ay
_4 re Sea se 4 aa <A %
eis b m
= fa < on Ba D < 99 aa ee Ox
a & 4 Za oe 2a iS Ba © FI Lae oe BO
RAT NUMBER < & ge a 8 z om BY S & A z eh iy
om a | fe |] 8e |] as | Ba a | oe PA Ae
eer aha ae Pee tes | as bee. leeal al
a a 4 qe Maciel iia
RSE Se | Si SEGAL ee | eo | cil jee) es
a Lon
as ZA Ga | ae | o& wi} of | Ha | on | oom 58
gl a a i a i) ao | a a

12 5/558 .4/570.9) 2.2 | 25.5/546 8/572 .3) 4.5 1143.2) 38.0) 3.3
15 .9/716 .3)732 .2) 2.2 | 25.8/833 .7/859 5) 3.0 |1591.7| 41.7) 2.6
1.4 | 22.2/645 .3/667.5) 3.3 |1295.8) 29.2) 2.3
541 .0/554.2) 2.4 | 20.1/615 8/635 .9) 3.2 |1190.1) 33.3) 2.8
1.7 | 12.9/672 .0/684.9) 1.9 |1848.9) 24.4) 1.8
2.3 | 28.0/602 .0/630.0) 4.4 |1412.6} 46.3} 3.3

© OOD ow bv
—
ies)
bo

Average.........| 13.4 655.4] 2.0 | 22.4 675.0) 3.3 |1830.4) 35.5) 2.7

* After deduction of nitrogen contained in same number of clean filter papers.

hot dilute acid, however, it would tend to offset to a small degree
the losses in the collection of urine, although the correction would
not be in proportion to the variable total urinary nitrogen. The
error in the collection of feces was found to be 3.3 per cent, using
the average of six determinations, or, comparing the total nitro-
gen lost to the total nitrogen excreted in urine and feces during
the week, the total error in the collection of both feces and urine
is 2.¢ per cent.

The results obtained in this test were applied to the metabolism
data for period 6, the period during which this test was conducted,

THE PROTEINS OF COTTONSEED MEAL 561

to determine what effect the incomplete collection of the ex-
creta has upon the utilization coefficients. It is evident that
an error in the collection of the excreta is reflected directly in
the nitrogen balance and in the percentage utilization. Using
the average values given in table 2 of 13.4 mgm. of nitrogen
representing uncollected urine and 22.4 mgm. of nitrogen repre-
senting uncollected urine and 22.4 mgm. of nitrogen representing
uncollected feces during a seven day period, and applying them
to the data for the individual animals during period 6 it is found
that the percentages of utilization of absorbed nitrogen as given
in the tables are approximately 2 per cent too high, while the
percentages of absorbed nitrogen retained are slightly more than
3 per cent too high. Like results are obtained for period 7
during which the animals received the same ration as in period 6.

The completeness of the collection of urine was tested in
still another way, that of the recovery of urea which was added
in the form of a standard solution to the daily feed. Two mature
rats were employed in the test. After the excretion of urinary
nitrogen had been reduced to a nearly constant level by sub-
sistence on protein free rations: for seven days, known amounts
of urea were added to the ration.

The excretion of this extra nitrogen was very prompt, as indi-
eated by the results of the test as shown in table 3. In the
case of rat 10, the results are somewhat difficult to imterpret,
owing to the fact that the consumption of feed decreased rapidly,
evidently resulting in catabolism of small amounts of body pro-
tein to furnish energy for the body. By using the figures for
the average excretion of nitrogen during the three days prelimi-
nary to the first urea day as the level of the endogenous nitro-
gen during the three urea cays, the apparent recovery of the
urea nitrogen amounted to 119 per cent. Similar results are
obtained if the average nitrogen excretion during the prelimi-
nary and subsequent periods areemployed. Ifit be assumed that,
owing to a decrease in feed consumption below that of the energy
requirements, the endogenous nitrogen should be taken as cor-
responding to that in the subsequent period, then the recovery
of the urea nitrogen was approximately 100 per cent. Undue

062 WwW. B. NEVENS

emphasis should not be placed upon the results given by this
animal, however.

With rat 11 more reliable data were obtained as it was not
evident that the feed consumption was deficient in meeting
the energy requirements. By using the average nitrogen excre-
tion during both preliminary and subsequent periods as the
level of the amount of body nitrogen excreted, the recovery of

TABLE 3

Test of the completeness of collection of urine by addition of urea to the ration

DAY LIVE WEIGHT FEED EATEN UREA N ADDED DAILY URINARY N
Rat 10
grams grams mgm. mgm.
1 182 10.5 0 35.6
2 10.5 0 24.0
3 10.5 0 23.5
4 178 6.1 42.1 74.6
5 6.1 42.1 83.0
6 6.1 42.1 76.0
7 4.6 0 35.9
8 167 5.6 0 35.8
Rat 11
1 176 or 0 42.8
2 Uo 0 30.2
3 ee, 0 39.8
4 Uoth 56.5 82.8
5 Call 56.5 101.8
6 5.4 0 BY
al 164 5.4 0 29.3

urea nitrogen amounted to 95 per cent of that fed. If the aver-
age of the amounts of nitrogen excreted during days 3 and 6 be
used as this level, then the recovery of urea nitrogen was prac-
tically 100 per cent.

It is evident from the data presented concerning the recovery
of urea nitrogen that the method for the collection of urine as
employed in these experiments, gives very nearly quantitative
results.

THE PROTEINS OF COTTONSEED MEAL 563

Further tests of the metabolism method employed, which were
performed in this laboratory and are described below, show that
loss of ammonia due to bacterial decomposition of the urine does
not occur to any appreciable extent.

In the first test, three portions of urine of 5 cc. each were
measured out for total nitrogen determination. At the same
time 5 ce. portions of urine were added to each of six metabolism
dishes containing the usual number of filter papers. ‘Three of
these dishes were allowed to stand at room temperature in the
metabolism laboratory, while the remaining three were placed
in an oven at a temperature of about 40°C. At the end of twenty-
four hours, the urine was collected from all six dishes in the same
manner as employed in the metabolism work, i.e., by washing
with hot acidified water. As a result of this test it was found
that 5 cc. of urine contained 28.18 mgm. of nitrogen, while the
amounts of nitrogen recovered from the dishes kept for twenty-
four hours at room temperature and at 40°C. were, respectively,
27.61 mgm. and 27.54 mgm.

In the second of these tests, the urine from one rat receiving a
constant amount of the same ration was collected daily. On the
first, third, and fifth days the urine was collected at once by
washing with acidified water in the usual manner. ‘The urine was
made up to a volume of 250 cc. and aliquots taken at once for
total nitrogen determinations.

On the alternate days the urine was not collected at the end of
the twenty-four hour period, but the filter paper was moistened
and the dish allowed to stand another twenty-four hours in the
metabolism laboratory before extraction in the usual maner.
The rat meanwhile was transferred to a clean dish. At the end
of the second day the urine was collected by washing as usual,
made up to volume, and aliquots taken for total nitrogen deter-
mination. The results of the test are shown in table 4. It is
evident that there was no appreciable loss of nitrogen due to
bacterial decomposition even after the metabolism dishes had
stood for two days.

How shall the nutritive value of proteins be compared? In
attempting to compare the biological values of various feeding-

564 W. B. NEVENS

stuffs, it is first necessary to select a suitable basis for compari-
son. Several different methods for comparison are in use. As
pointed out in the introduction one of the most common methods
is to base conclusions upon the character of the growth secured,
the principal index in such a case being the gain in live weight.
Some of the data from table 1 of the appendix are brought
together in table 5. These data were all obtained in periods 2
and 8. The rats consuming the cottonseed meal ration showed
marked fluctuations in gain in live weight which can not be ac-
counted for on the basis of a variable food intake. With the
corn ration, there was a gain in weight by one rat in one period

TABLE 4

Effect of allowing metabolism dishes to stand twenty-four hours and forty-eight
hours before the collection of urine

DAILY URINARY N wHEN DAILY URINARY N wHEN
DAY OF EXPERIMENT COLLECTED AT END OF TWENTY- COLLECTED AT END OF FORTY-
FOUR HOURS EIGHT HOURS
mgm. mgm.
1 57.9
2 64.9
3° 60.8
4 67.3
5 63.2
6 60.3
ANVETAZE Mh ei sume ee 60.3 62.1

only. The nitrogen of the ration, however, was being used by
the body to a considerable extent, for on a nitrogen-free ration
the same animals lost 16 to 19 grams in weight during a period
of equal length, compared to 1 to 2 grams on the corn ration.
Likewise, there was also a large variation in gains in weight by
the rats receiving the alfalfa ration, the average gains of rats
7 and 8 being almost zero. It is probable that many factors
other than the quality and amount of the protein consumed
influence the gain in weight, such as the proportion of carbohy-
drates in the ration, amount of water drank, exercise, the pro-
portions of gain which is protein or fat, etc. While interpreta-
tions based upon the gain in live weight may lead to reliable

THE PROTEINS OF COTTONSEED MEAL 565

conclusions in some instances, the adoption of such a criterion
in the present case would certainly be a fallacious procedure.

It is a matter of common knowledge that all animals require
protein food for keeping the body tissues intact, known as the
maintenance requirement, and secondly, that growing animals
need an additional quantity of protein for the construction
of new tissue. If a standard for the comparison of the value of
the proteins of feedingstuffs for growth is based simply upon the
proportion of the nitrogen of the feedingstuff which is retained
by the body, the values secured in such a manner are subject
to gross errors. With such a method of computation the ap-
parent value of the proteins for growth depends largely upon the
nitrogen intake, or, in other words, upon the amount of feed
eaten, and this in turn is subject to individual idiosyncracy and
the palatability of the ration. As mentioned elsewhere, when
the ration proves unsatisfactory, rats tend to eat less and less
from day to day. By reference to table 5 it may be seen that
both rats 5 and 6 ate less of the corn ration during period 3
than during period 2. Rat 5, during period 2, retained 16 per
cent of the nitrogen absorbed, but during period 3, when the
amount of feed consumed was evidently too little to maintain
the animal’s live weight, the nitrogen of the excreta was greater
than the nitrogen intake, so that there was a loss of nitrogen from
the body resulting in a negative value for the percentage of
absorbed nitrogen retained. Similarly, the percentage of ab-
sorbed nitrogen retained by rat 6 falls from 23 per cent in period
2 to 9 per cent in period 3, a change which in this instance may
also be attributed to a decreased food intake. Were the average
percentage of the absorbed nitrogen retained by rats 5 and 6
taken as a measure of the utilization of the proteins of corn for
growth, it would be a distorted picture of the facts.

There seem to be factors other than the amount of feed con-
sumed which render the use of the percentage of absorbed nitro-
gen retained an unsatisfactory criterion of the utilization value of
the proteins of feedingstuffs. As may be seen by reference to
table 5, the percentage of nitrogen retained by rat 7 in periods
2 and 3 falls from 23 per cent to 8 per cent, and in the case of

566 W. B. NEVENS

rat 8 the percentage falls from 16 per cent in period 2 to 6 per
cent in period 3. These violent fluctuations are not due entirely
to a decreased nitrogen intake, for in the case of rat 7 the
feed intake increased slightly during the second period. They
are, however, associated with a slightly decreased digestibility,
although there may be other causative factors.

TABLE 5
A comparison of three methods of expressing the utilization of proteins

UTILIZA-

GAIN TION OF

PERIOD
el
gm. gm. per cent per cent

1 2 Cottonseed meal 9.52 11 63 31
1 3 Cottonseed meal 9.48 6 65 29
2 2 Cottonseed meal 8.66 9 64 21
2 3 Cottonseed meal 8.57 5 64 17
3 2 Cottonseed meal 11.51 12 71 44
3 3 Cottonseed meal 12.47 11 70 43
4. 2 Corn 7.44 2 49 15
4 3 Corn 8.05 0 47 15
5 2 Corn 7.66 0 54 16
5 3 Corn 6.30 —1 43

6 2 Corn 7.49 —1 55 23
6 3 Corn 6.35 —2 48 9
7 2 Alfalfa hay 9.33 6 62 23
7 3 Alfalfa hay 9.50 —5 57 8
8 2 Alfalfa hay 9.19 2 58 16
8 3 Alfalfa hay 8.20 —2 57 6
9 2 Alfalfa hay 11.42 3 67 22
9. 3 Alfalfa hay 13.67 7 73 38

* For method of calculation of these percentages, see table 1 of the appendix.

Any suitable criterion used in feeding experiments for the
comparison of the utilization of proteins for growing animals
must necessarily consider the effect of the proteins in providing
nitrogen for maintenance, for in growing animals these processes
proceed concurrently. It is doubtful if the true protein require-
ment for the maintenance of a growing animal can be determined
by feeding a ration containing protein, for Waters (23) has shown

THE PROTEINS OF COTTONSEED MEAL 567

that when young steers received a ration which just maintained
their live weight some of the growth processes continued. Simi-
lar results were obtained by Aron (24).

Perhaps the nearest approach to the determination of the
exact amount of nitrogen required for the maintenance of a
growing animal is a study of the nitrogen excretion when the
ration consists entirely of carbohydrates and this is being taken
in an amount in excess of the body’s energy requirement. Under
such conditions the nitrogen excretion falls to a very low level,
often to one third or less of that during starvation, as shown by
Folin (25), Landergren (26), Cathcart (27) and Thomas (28).
The amount of protein then being catabolized has been defined by
Rubner (29) as the ‘‘ wear and tear”’ quota of protein metabolism,
which requires a ‘‘repair quota”’ of protein in the diet in order
to replace it. A ‘“‘growth quota’’ must be supplied the young
animal in addition to the ‘‘repair quota”’ in order that growth
may take place. Using dogs as experimental animals, Michaud
(30) found, when protein in the form of casein or dog tissue
was fed in amounts equivalent to the protein minimum after
the metabolism had been reduced to this level, that there was
no further loss of nitrogen from the body. Thomas (28) found,
after the reduction of the nitrogen excretion by a carbohy-
drate diet to the minimum level, that nitrogen equilibrium could
be restored by the ingestion of an amount of protein nitrogen
in the diet equal to the amount of nitrogen being eliminated in
the excreta.

On a nitrogen-free diet the amount of nitrogen excreted daily in
the feces was about 1 gram, and this amount was not increased
with a nitrogen intake of 3 grams furnished by a highly diges-
tible protein. With diets producing a large bulk of feces he
found that a greater proportion of digestive juices was elimi-
nated, increasing the nitrogen content of the feces.

In the interpretation of the feeding experiments which follow
it is assumed that the amount of protein required for body main-
tenance is a constant value for each individual at a given weight.
Such an assumption is entirely in harmony with the theories of
many investigators in the fields of both human and animal nu-

568 W. B. NEVENS

trition. Folin (25), as a result of his study of the different forms
in which nitrogen is excreted on high and low protein diets, was
led to formulate his theory of two distinct types of metabolism.
The endogenous is most characteristically represented by the
excretion of creatinine, which, ‘‘on a meat-free diet is a constant
quantity, different for different individuals, but wholly inde-
pendent of quantitative changes in the total amount of nitrogen
eliminated.”’ Folin’s results have been substantiated by an
immense amount of investigation concerning urinary creatinine,
and his theory of protein metabolism is now almost universally
accepted in its main essentials, although it has been necessary
to modify this view slightly with our increased knowledge of the
chemistry of the proteins.

The constancy of the protein minimum for the individual is
accepted by Lusk, Thomas and others. That this minimum
differs between individuals and is subject to slight variation due
to environmental, temperamental and dietary changes, possi-
bilities which are not precluded by Folin’s theory, is brought out |
by Cathcart (81):

As regards the uniformity of the protein minimum it may be defi-
nitely stated that there is no single minimum—common to all men and
to all conditions. Rubner, Caspari and others also hold firmly to this
opinion. Caspari quotes the work of Larguier des Bancels in 1903
in confirmation of this belief in the existence of mutiple protein minima.
The facts that can be cited against a common minimum are many in
number. Thus the caloric value of the diet given influences very
materially the amount of nitrogenous material required, as is shown,
for example, in the experiments of Voit and Korkunoff. Then, as
Rubner has pointed out, the temperature influences quite markedly
the course of protein metabolism. Finally, another factor of consid-
erable importance may be mentioned, the activity of the organism.

In the sphere of animal nutrition, the constancy of the main-
tenance requirement for farm animals is recognized by Kellner,
Armsby and Haecker. C. Voit and Kellner also proved conclu-
sively that work production of varying intensity by farm animals
does not increase the protein metabolism appreciably.

THE PROTEINS OF COTTONSEED MEAL 569

In this connection it should be stated that some of the cur-
rent theories of protein metabolism are not in complete harmony
with that just mentioned. Among these are the reversible reac-
tion theory of Sherman (32), which seeks to account for the func-
tions which the food protein serves in body maintenance by the

_assumption that the absorption of the amino acids liberated in

digestion causes an increased concentration of these in the tis-
sues which checks or even reverses the hydrolysis of tissue protein.
This theory is hardly compatible with the known facts regarding
the constancy of the endogenous metabolism, which has been
found (83) to be uniform from hour to hour, as evidenced by the
creatinine elimination, even during digestion and absorption of
proteins. Absorption of the protein digestion products from the
alimentary tract presumably occupies only a portion of the
twenty-four hour period, so that even if the endogenous catab-
olism were inhibited by the increased concentration of amino
acids, it would be only temporary, for it has been shown (34)
that, in adult rats, protein feeding has only a very slight effect
upon the amino acid concentration in the tissues. This known
slight increase in the amino acid content of the tissues during
digestion would not be of sufficient magnitude to inhibit the
action of digestive enzymes when a digestion experiment is
conducted in vitro. Further, it is unreasonable to assume that
anabolism and catabolism of tissue proteins are simply rever-
sible phases of the same reaction and that both these processes
are promoted by the same enzyme. In the young growing
animal protein feeding has been demonstrated (384) to increase
considerably the amino acid content in the tissues. Were the
endogenous metabolism inhibited entirely during the time this
concentration is maintained, as must be assumed from the re-
versible reaction theory, then the catabolism of tissue protein
per unit of weight in the young growing animal would be but a
fraction of that of a mature animal.

Osborne and Mendel (35) explain the maintenance protein
requirement upon the need of certain amino acids to serve special
physiological functions, such as the formation of the active prin-
ciples of the internal secretions and hormones. ‘This theory

570 W. B. NEVENS

assumes, therefore, that when the animal is receiving a nitrogen-
free ration, body tissue must be catabolized to furnish the essen-
tial amino acids, but that, on the other hand, when a ration con-
taining a complete assortment of amino acids in sufficient amount
is being consumed the endogenous metabolism is only a frac-
tion of that on a non-nitrogenous ration, and that then only
the catabolism of the internal secretions or the tissues which
regulate metabolism would be affected. Under these condi-
tions the muscles would scarcely be affected and the creatinine
elimination would bear little relation to the endogenous metab-
olism. Moreover, the theory does not satisfactorily account
for the effect of ammonium salts, mixtures of amino acids and
single amino acids in partially supplying nitrogen for main-
tenance.

Since the plan of procedure and method of calculation employed
in this investigation are dependent primarily upon the basic
assumption that the endogenous metabolism of the animal or-
ganism is constant in character and amount for an individual
at a given age and weight, an examination of the data obtained
during all the metabolism periods was made in order to ascer-
tain, if possible, whether this assumption is substantiated by
the experimental results at hand. In making this examination,
the data embodied in appendix table 1 were employed to obtain
the first set of values shown in the column headed ‘‘As deter-
mined’’ under each ‘‘period”’ of table 6. These values were
obtained by deducting the sum of the endogenous nitrogen and
the metabolic nitrogen in the feces from the daily urinary nitro-
gen and calculating the percentage of the daily nitrogen intake
which the remainder forms. The second column of values under
each ‘‘period,’’ headed ‘‘ As calculated, ’’ was obtained in the same
way as those in the first column, except that the average value
for daily urinary nitrogen as determined with nine rats in period
1, i.e., 22 mgm. per 100 grams live weight, is used in calculating
the ‘‘endogenous nitrogen” for periods 2 to 7, inclusive, instead
of the individual values determined in the same period.

It is shown in table 6 that the individual daily urinary nitrogen
values for rats 2,5, and 9 are above the average, while those for

THE PROTEINS OF COTTONSEED MEAL 571

rats 3, 6 and 8 are below the average. Hence, for comparison,
rats 2 and 3, 5 and 6, and 8 and 9 are ommemned | in pairs. The
rats of each pair sacalvedl the same rations throughout the ex-
periment. It is natural to assume that two animals of the
same age and weight and in a comparable nutritive condition
will utilize the same ration with an equal degree of efficiency,
subject of course to inherent individual variability. By refer-
ence to table 6, it may be noted that this holds true to a very
great extent, albanien the natural variations to be expacieel 1 in ~
biological work of this kind are in evidence.

TABLE 6

The proportion of the daily intake of nitrogen above maintenance which appears
in the urine (expressed in percentage)

PERIOD 2 PERIOD 3 PERIOD 4 PERIOD 5 PERIOD 6 PERIOD 7

ee eee Agia UAsil VAs Agel Asi. Agi i|) As.) Agi! Ag |/As. |) Ag il) cAs
deter-| calcu-| deter-| calcu-} deter-| calcu-| deter-| calcu-} deter-| calcu-| deter-| calcu-

mined] lated |mined|] lated |mined] lated |mined] lated |mined] lated |mined| lated

2 12.0 16.3 | 9.8 |13.1 7 | 5.3 | 8.3 |12.4 | 8.9 |13.4 | 8.8 |13.7
3 SESH Oro 9 Ae S-Ouilie Sulla)! @.d 16-9 18929e! 9231 79):.9) | 952

NH

5 26.5 |29.1 |35.0 |38.2 |18.7 21.0 |18.5 |21.1 |12.3 |14.4 |11.4 |13.7
6 23.9 |20.0 31.3 |26.7 |21.0 17.5 |16.4 {12.3 |12.6 | 9.5 14.0 |11.1

6 (14.3 |10.4 | 8.9 |14.3 |12.5 | 9.7
8 {1 6.4

7.7
7.3 {14.9 |16.4 |16.0 |17.6 |16.4 18.1

18.

Considering first the values listed under the headings ‘‘As
determined”’ in each period it is evident that there is a marked
uniformity exhibited by the animals of each pair throughout the
different experimental periods with but few exceptions. For
example, it is shown that rats 2 and 3 eliminate in the urine
about the same proportion of the nitrogen intake above main-
tenance ‘‘as determined,”’ with the exception of periods 2 and
4. Rats 5 and 6 show quite uniform results throughout the
entire six periods. Rats 8 and 9 do not very widely from each
other in periods 4, 5 and 6. Moreover, certain rations seem to
have a pronounced effect on these percentages. Rats 5 and 6
received the corn ration during periods 2 and 3, and the corn-

572 W. B. NEVENS

cottonseed meal ration during periods 4 and 5. With both of
these rations, the proportion of the daily nitrogen intake elimi-
nated in the urine was greater than with the other rations, but
both animals behaved alike in this respect.

On the other hand, when the average values for the endoge-
nous nitrogen of the urine are used in calculating maintenance, the
variations in the percentages of the nitrogen intake above main-
tenance appearing in the urine of the animals of each pair are
greatly exaggerated in 14 of the 18 cases involved, as shown under
the headings ‘‘As calculated.’’ In one of the four cases, that of
rats 5 and 6, period 7, there is no change. In the other three
cases, those of rats 2 and 3, period 4, and rats 8 and 9, periods |
2 and 3, the spread is lessened slightly. In many cases the use
of an average maintenance factor increases the spread between
the animals of a pair as much as 200 per cent. These data seem
to indicate quite conclusively that the endogenous metabolism
is a function of the individual animal and that this is a definite
and probably constant value under a given set of conditions.
This is quite in harmony with the theory of Folin (25) respecting
the constancy of endogenous metabolism. In calculating the
results of these experiments, therefore, the use of individual
maintenance values is evidently justifiable.

If, having determined the minimal ‘‘wear and tear’’ quota of
an animal by appropriate metabolism experiments, feeding
tests are then initiated to study the utilization of the proteins of
feedingstuffs by that animal, it is possible to calculate the pro-
portion of the nitrogen excreted in the urine which is of endoge-
nous origin and likewise the amount of fecal nitrogen whose source
is metabolic. ‘This method follows closely that of Thomas (28)
in calculating the biological values of foodstuffs. Such a criterion
evaluates the proportion of the nitrogen of the food which is
actually utilized by the animal in its metabolism, whether the
animal is consuming an amount of protein which is not quite
sufficient for it to maintain its live weight, or whether growth is
permitted. The values obtained by applying this method of
calculation to the data of metabolism periods 2 and 3 are shown
in the last column of table 5. An inspection of these values

THE PROTEINS OF COTTONSEED MEAL 573

shows that this method overcomes some of the objections raised
to the other methods. When the gain in live weight falls to
zero or a little below, but at the same time it is evident that some
of the nitrogen of the ration is being used by the animal, the
percentage ‘“‘utilization of the absorbed nitrogen for mainte-
nance and growth” is lowered, but is, nevertheless, a very dis-
tinct positive value. With a slight decrease in food intake, there
is usually a corresponding fall in the ‘‘utilization of absorbed
nitrogen for maintenance and growth”’’ coefficient, but this
decrease is not so extreme as when the results are calculated upon
the basis of the ‘‘absorbed nitrogen retained.’”’ Moreover, a
decrease in the feed intake to just below the maintenance level
does not result in a negative value. On the whole the ‘‘utiliza-
tion of absorbed nitrogen for maintenance and growth” coeffi-
cients are much less variable than those of the ‘‘absorbed nitro-
gen retained.”’ ‘The former method is subject to a coefficient
of variability of 8.4 per cent when all of the values obtained in
the six metabolism periods are considered, and a mean is assumed
for each ration. Similarly, the same values when calculated
upon the basis of the percentage of ‘‘ absorbed nitrogen retained ”’
have a coefficient of variability of 27.7 per cent, a striking and
important difference.

In this investigation, therefore, the endogenous metabolism
of the experimental animals was studied during a metabolism
period in which a nitrogen-free ration was fed, and this was fol-
lowed by six metabolism periods in which the proteins of cot-
tonseed meal were compared with those of corn and alfalfa hay.

IiI. DISCUSSION OF THE RESULTS

Metabolism of the rat on a nitrogen-free ration. During the
first eleven days of the experiment the rats which had been con-
suming an ordinary stock ration were given nitrogen-free rations
prepared as described above. On the fifth day collection of the
feces and urine was begun, and continued for seven days. In
order to check the results secured during the first period of the
experiment, three of the rats were again placed on nitrogen-free

574. W. B. NEVENS

rations during period 6, after having received nitrogenous rations
during the intervening time. The principal data of these trials
are included in table 7. The rats lost slightly more than 2 grams
of weight per head daily. For the first few days of the period the
animals ate the ration in large quantities, but as they apparently
found the feed unsatisfactory, they consumed smaller and smaller

TABLE 7

Metabolism of the rat when receiving a nitrogen free ration

DAILY
FECAL
ANRRINSH | ying painy | URINARY | virRoGEN
RAT TOT INITIAL FINAL FEED ES VAGUA, PER a
NUMBER WHIGHT | WEIGHT |CONSUMED|. op ogmn | NITROGEN 100 Grams I) Gane
DAILY LI —
WEIGHT

grams grams grams mgm. mgm mgm mgm

1 1 99 88 6.51 25 17 27 253

2 1 118 102 7.43 3l 19 28 255

3 1 134 116 9.07 26 24 21 259

4. i 135 118 8.79 24 22 19 251

5 1 120 104 7.29 28 20 25 273

6 1 129 110 7.86 21 Dili 18 270

7 1 123 107 7.43 23 20 20 266

8 1 122 109 8.14 22 24 19 298

9 1 126 114 7.43 29 18 25 246
Average 7.78 22 264
1 6 113 102 6.10 22 19 20 306

4 6 135 130 9.02 16 20 15 223

0 6 118 105 4.76 24 17 21 357
Average 6.63 19 295

amounts from day to day, and a few of the animals scattered the
feed from the containers at once upon being fed.

It was found that, while subject to some individual variation,
the amount of urinary nitrogen per 100 grams of live weight is
fairly constant, the average value of 22.4 mgm. obtained agreeing
almost exactly with that found by Mitchell (20) in a large num-
ber of metabolism periods. From the results secured in period
6 it appears that there is a slightly lessintense endogenous metab-
olism as the animal becomes older, as evidenced by a decreased

THE PROTEINS OF COTTONSEED MEAL 575

excretion of urinary nitrogen per 100 grams live weight. The
slight increase in the case of rat 7 during period 6 as compared
with period 1 is evidently due to a deficient food consumption
during the former period, necessitating the catabolism of body
protein to furnish energy. The individual values obtained in
period 1 for urinary nitrogen per 100 grams live weight are used
in the subsequent tables for calculating the utilization of the
various rations, the values always being corrected to the average
live weight of the particular animal during that period.

The quantity of fecal nitrogen per 100 grams feed when the
rat is consuming a nitrogen-free ration is quite a constant factor,
although this relationship seems to be affected somewhat by ex-
tremes in the amount of feed consumed, as may be noted in the
case of rats 4 and 7, period 6. Perhaps a more potent factor in
causing this fluctuation is the varying amount of filter paper
eaten, as found by Mitchell (20) in an experiment in this labora-
tory in which rats during one period had no access to filter paper
and during the other actually consumed some paper.

It is recognized that the calculation of the metabolic nitrogen
in the feces by the method described is subject to an error when
applied to a variety of rations. Were the content of crude fiber
in all rations the same as in the synthetic nitrogen-free ration,
the assumption that the metabolic nitrogen of the feces varies
directly with the amount of feed consumed would be valid, but
with rations varying as widely in the percentage of crude fiber
as the corn and alfalfa rations, the adoption of such an assump-
tion evidently leads to an error of undetermined magnitude.
However, the method of correcting the absorbed nitrogen and the
nitrogen balance, by the use of the factor for metabolic fecal
nitrogen obtained on nitrogen-free rations, undoubtedly gives
values nearer the truth than if no such corrections were made,
since the actual metabolic fecal nitrogen on the experimental
rations containing protein was very probably greater per 100
grams of food consumed than the factors used. In computing
the amount of metabolic fecal nitrogen shown in the tables that
follow, the values for fecal nitrogen per 100 grams feed eaten in

576 W. B. NEVENS

period 1 in the case of each animal are applied to the data for the
same animals in later periods.? :

If it is true, as seems probable from the meager data obtained,
that the endogenous metabolism of the rat becomes less intense
per unit of live weight as the animal approaches maturity, then
in conducting investigations of the kind under consideration it
would, no doubt, be advisable to introduce a metabolism period
using a nitrogen free ration every six or eight weeks. With these
data available it would be possible to make linear corrections for
any changes in the requirement of the basal metabolism. In
the tables showing the utilization of the proteins which follow,
such corrections have not been attempted with the limited num-
ber of data at hand, but should the data secured with rats 1 and 4
be used for such a purpose, it would necessitate changes in the
utilization coefficients given of not more than 1 to 2 per cent
as a maximum.

Palatability of rations. The results obtained in the six metab-
olism periods during which nitrogenous rations were fed are
summarized in table 8.

From an inspection of the figures giving the amounts of feed
consumed daily it is evident that all rations containing cotton-
seed meal were readily consumed by the animals, attesting to
the palatability of this feed, even when forming as much as 24
per cent of the ration. When corn was the sole source of protein
in the ration the amounts of feed consumed were smaller than
with any other ration. Ground corn seems to be less palatable
to rats than whole corn for the latter is usually eaten readily.
Perhaps another reason why less of the corn ration was consumed
than the alfalfa ration, for example, is that the corn ration was
much more digestible and had a higher calorific value, so that
less of it was required to supply the energy requirements. ‘The
ration in which cottonseed meal and corn were combined was con-
sumed in greater quantities than the corn ration but not so freely
as the cottonseed meal ration. Alfalfa hay proved very palatable,
as all rations of which it formed a part were readily eaten.

2 These values, as well as those for urinary nitrogen, when being used for these
calculations, were extended to one more decimal place than shown in table 7.

TABLE 8

The utilization of the proteins of cottonseed meal, corn
summary of results

RAT
NUMBER

il

2

3
Average

1
2
3

Average >

2
3
Average

4

5

5
Average

4

5

6
Average

5
6
Average

7

8

9
Average

a

8

9
Average

8
9
Average

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RATION

Cottonseed meal

Cottonseed meal +
alfalfa hay

Cottonseed meal +

alfalfa hay + corn

Corn

Cottonseed meal +
corn

Cottonseed meal +
alfalfa hay + corn

Alfalfa hay

Alfalfa hay + corn

Alfalfa hay ++ corn

+ cottonseed meal

577

AVERAGE LIVE
WEIGHT

grams

98
109
127
111

114
126
159
133

136
186
161

118
108
115
114

129
118
124
124

139
141
140

104
103
121
109

115
118
134
122

142
154
148

TROGEN

FEED CONSUMED
DAILY

ABSORBED NI-

grams| mgm.

9.50/124.8
8 .62/107.1
11.99)169.6
10.04/133.8

10.83)125.9
11 .06/131.7
15 .68)185 .0
12 .52)147.5

9 .82)124 .3
14.01)173.0
11.92}148 .7

7.75}118.9
6 .93}105 .9
6 .92/104.5
7.22/109.8

9.81/141.4
8.30)127.8
8.11/120.2
8.74/129.8

10.90/136.9
11 .20/146.8
11 .05)141.9

9.42) 99.4
8.69} 92.9
12 .55)128.1
10.22/106.8

12 .64/149.1
14.08]175.8
12.51/166.6
13 .038)163 .8

11 .81/152.6
13 .06/184.0
12 .83)168 .3

and alfalfa hay;

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38.2) 30.4 64] 19
91.3) 28.0, 71) 44
61.3) 27.9) 66] 31
02.9) 30.6) 67 | 26
58.1) 35.7) 71} 29
89.5) 33.7) 67) 34
66.8) 33.3) 68] 29
45.6} 38.2} 63] 21
72.5) 39.4, 65 | 27
59.0) 38.8) 64] 24
34.6) 22.8) 48) 15
24.7) 27.1; 49 8
34.1) 20.3) 52] 16
31.1) 23.44 49} 13
59.5) 24.9) 60) 30
43 .6| 29.2) 59] 21
48 .9) 22.3) 60] 28
50.7) 25.5) 60] 26
49.3) 34.9) 62) 18
64.8) 25.1) 61) 28
57.1) 30.0, 61) 23
37.2) 21.4, 60) 16
33.7/ 20.0) 58] 11
61.2) 29.4, 70} 30
44.0) 23.6) 62] 19
56.8) 23.3) 54} 20
78.9) 23.3) 59 | 28
70.6} 32.7) 62) 29
68.8] 26.4, 58 | 26
65.0) 27.0| 61) 26
76.1) 38.0} 62) 29
70.6, 33.0) 61 7

578 W. B. NEVENS

Utilization of proteins. In the summary of results shown in
table 8 two methods of calculating the utilization of the proteins
fed are included for comparison, although, for reasons discussed
above, the second method, namely, the utilization of the absorbed
nitrogen for both maintenance and growth, is employed i in the
discussion here.

The utilization of the nitrogen absorbed from a cottonseed
meal ration containing 10 per cent of crude protein was found to
be 66 per cent, using the average results of six metabolism
periods with three rats. The utilization of the proteins of
alfalfa hay was found to be only slightly less than that of
cottonseed meal, namely, 62 per cent.

When these two feeds were combined in such proportion that
each furnished about an equal amount of digestible protein to
the ration, very interesting results were secured, indicating a
slight supplementary effect of the proteins from these two sources.
This effect was not pronounced, the utilization percentage
being 2 per cent above that of cottonseed meal alone and 6 per
cent above that of alfalfa hay alone. It is noted that during the
periods when the cottonseed meal alfalfa hay ration was fed,
greater quantities of feed were consumed and larger amounts of
nitrogen were absorbed than with either the cottonseed or
alfalfa hay rations alone, which may in some unknown way have
operated in effecting a more efficient utilization of the nitrogen,
although the same conditions hold true in the case of both groups
of rats which received either the corn or the alfalfa ration during
two periods and were then changed to rations containing proteins
from both sources.

It was found that the proteins of corn were wtilheed the least
efficiently of those of the three feedingstuffs compared. When
corn was combined with cottonseed meal or with alfalfa hay the
resulting utilization coefficients tended toward a mean of the
utilization coefficients secured with these feedingstuffis when
fed alone, but were nearer that of the feed other than corn. For
example, the utilization coefficients found for the corn and
cottonseed meal rations were 49 per cent and 66 per cent
respectively, the mean of these two being 57.5 per cent, but

ee

THE PROTEINS OF COTTONSEED MEAL 579

the utilization coefficient found for the cottonseed meal-corn
ration was 60 per cent. Possibly this represents a slight
supplementary relationship.

The results obtained with the ration in which cottonseed
meal, corn and alfalfa hay were combined were remarkably
uniform. Of the twelve values obtained with rats receiving this
ration during two metabolism periods each, the lowest value was
57 per cent and the highest 67 per cent, the average of all being
63 per cent. The combination of the proteins from three dif-
ferent sources failed to indicate any farther supplementary effect
of the proteins.

The high nutritive value of the proteins of cottonseed meal
manifested by these experiments is in substantial accord with
the conclusions of Richardson and Green (1), Osborne and
Mendel (3, 4, 5, 6) and McCollum and Simmonds (6). They do
not seem to be in harmony with the findings of Hart and Hum-
phrey (8) who studied the utilization of the proteins of cottonseed
meal for milk production, but since growth and milk production
are dissimilar functions an absolute comparison of the results of
the two experiments is not valid.

Correlation of chemical composition with nutritive value. In
seeking for an explanation of the differences in the nutritive value
of the proteins of these feedingstuffs based upon differences in
their chemical makeup, it is evident first of all that their nutri-
tive values do not vary so widely as the analytical data at hand
would indicate. For example, the differences found between
the utilization of the proteins of cottonseed meal and alfalfa
hay was but 4 per cent,while from an examination of the data
in table 5 of a preceding paper, it is apparent that cottonseed
meal contains more than twice as much arginine nitrogen and
nearly twice as much histidine nitrogen as alfalfa hay, while the
latter contains more than three times as much nonprotein nitro-
gen as the former.

Several theories may be advanced in explanation of this ap-
parent inconsistency. In the first place, alfalfa hay is shown
to have a lower digestibility than either cottonseed meal or corn.
There is no evidence to preclude the possibility that the char-

580 — W. B. NEVENS

acter of the nitrogen absorbed from alfalfa hay differs qualita-
tively from that remaining in the undigested residues. Judging
from the ease with which tyrosine is split off from proteins in
tryptic digestion in vitro, it is possible that the absorbed nitro-
gen contains a greater proportion of amino acids essential to the
body than the unabsorbed portion. Further, the sterochemical
arrangement of the amino acids in the protein molecule may
affect the extent to which the digestive enzymes are able to
cause hydrolysis of the different proteins. A second considera-
tion is the possible interchangeability of the various forms of
nitrogen in nutrition, as already pointed out in the case of arginine
and histidine. To what extent the nonprotein nitrogen of alfalfa
hay is utilized in maintenance is problematical, but since there is

no reason to doubt that the degradation products of crude

protein are able to serve in this capacity, it is possible that a large
part of the absorbed nonprotein nitrogen fulfills some of the
requirements of the animal body.

It is reasonable to assign the higher content of the basic amino
acids of cottonseed meal as the reason for its superiority over the
proteins of alfalfa and corn. In the case of the last mentioned
feedingstuff, there is the additional factor of a comparatively
low lysine content to be considered, although from the studies
of Osborne and Mendel concerning the lysine requirements
for growth, a lysine content of 2.2 per cent of the protein oe
to be ample for normal growth.

In the absence of further information respecting the char-
acter of the mono-amino acid and nonprotein nitrogen content
of these feedingstuffs, a detailed picture of which the Van Slyke
analysis does not include, correlations between the chemical
composition and nutritive value of the proteins of feedingstuffs
can proceed little beyond the realm of the functions and rela-
tionships of the basic amino acids.

Comparison of feed consumption with that of farm animals. It
was noted during the course of this investigation that the rats

consumed an enormous amount of feed in proportion to their

live weights. In some few cases the amount of air dry feed
eaten daily was equivalent to as much as 10 or 11 per cent of the

ee

TABLE 9

Comparison of feed consumption by albino rats with that of farm animals

NUM- ay- | EATEN
SPECIES OR BREED LENGTH BER OF AV- DAILY
AND OF FEEDING FEEDS IN RATION ANI- | ERAGE eae PER 100
CLASS OF ANIMAL PERIOD MALS AGE WEIGHT POUNDS
FED LIVE
WEIGHT
days days | pounds | pounds
28 Corn + oats + alfalfa | 10 227 | 854 | 2.2
Percheron hay
hilliess: yee 28 Corn + oats + alfalfa 10 683 |1484 | 1.8
hay
35 Corn + linseed meal + 4. 978 | 2.5
Hereford clover hay
steerst 28 Corn + linseed meal + 4 1466 | 1.5

clover hay

30 Corn + bran + linseed 4 365 | 472 | 2.9

$ oil meal + alfalfa hay
Ueesey ne Mens 90 Corn + bran + linseed 4, 730 | 889 | 1.8
oil meal + alfalfa hay
30 Corn + bran + linseed 4 365 | 656 | 2.4
Holstein oil meal + alfalfa hay
heiferst..... 90 Corn + bran + linseed 4 730 |1112 | 1.8
oil meal + alfalfa hay
174 38 | 6.0
Swine§........ 495 128 | 3.8
105 320 | 2.4
45 ell
Sheeprmage acne 127 11.1
42 Cottonseed meal+corn| 9 129 {| 8.48§
+ alfalfa hay + syn-
Albinorats ... thetic mixture
21 or 18 per cent protein 17 175 | 4.68§
more*** | 20077)

* From Bul. 192, ill. Agr. Exp. Sta.

+ From Bul. 197, Ill. Agr. Exp. Sta. Data concerning ‘‘Full-feed lot.”’

t From Nebr. Agr. Exp. Sta. Unpublished manuscript. Data concerning
“Heavy fed groups.”

§ From Henry and Morrison, Feeds and Feeding, 15th ed. p. 569.

** Calculated from data of Weiske as quoted by Armsby, The Nutrition of
Farm Animals, p. 432.

tT Grams.

§§ Grams per 100 grams live weight.

*** From Osborne and Mendel. Protein Minima for Maintenance. Jour.
Biol. Chem., 1915, xxii, 241.

581

582 W. B. NEVENS

live weight. It seemed of interest to compare the feed consump-
tion of albino rats with that of farm animals. Such a comparison
is made in table 9. The tabulations of horses and cattle include
in each case two entries of the same group of animals at dif-
ferent ages and weights. It is known that the horses and cattle
were restricted in the amount of concentrates consumed but were
offered roughage to practically the limit of their appetites. Hence
a comparison of the feed consumption of these animals with that
of the first group of rats, which were the ones concerned in this
investigation, is warranted, but the data are indicative only.
Data for the amount of feed consumed by the swine, sheep and
second group of rats is not at hand.

It is evident from the data presented that the rat is a voracious
eater, even when receiving rations comparable to those of farm
animals. A rough approximation places the relative amounts
of feed eaten by rats as about three times that of various breeds
and classes of farm animals, if swine be excepted. ‘The fact is
also brought out, as has previously been noted by others, that
the young animal consumes much more feed in proportion to
live weight than when older and heavier.

Cottonseed meal probably not toxic to albino rats. None of the
rations containing cottonseed meal seemed to exert any harmful
influence upon the rats consuming it. ‘Three of the animals
received continuously for 7 weeks rations containing from 7.7
per cent to 23.9 per cent cottonseed meal with no evidence of
toxic symptoms but remained in excellent nutritive condition.
This observation is in agreement with those of Richardson and
Green (1) and Osborne and Mendel (5).

SUMMARY OF THE DISCUSSION OF THE NUTRITIVE VALUE
OF THE PROTEINS

Evidence is presented to show that metabolism experiments
with the rat as a subject can be carried out with a high degree
of accuracy.

Different methods of expressing the nutritive value of the
proteins of feedingstuffs are discussed. The plan of employing the

f
|

THE PROTEINS OF COTTONSEED MEAL 583

results secured in a preliminary and final metabolism period
during which the animal receives a nitrogen free ration, for the
calculation of the percentage of the absorbed nitrogen utilized,
is favored. In comparing the nutritive value of the combined
proteins of the feedingstuffs cottonseed meal, alfalfa hay and corn,
it was found that when one of these feeds furnished the sole
source of protein in rations containing 10 per cent of crude pro-
tein, the utilization of the proteins for the growth of albino rats
was, in the order in which the feedingstuffs are named, 66 per
cent, 62 per cent and 49 per cent, respectively.

When rations containing these feedingstuffs, combined in
various ways, but with each feed furnishing an equal amount of
digestible protein, were fed, there was evident no clear cut sup-
plementary effect of the proteins of one feed upon another,
except in the case of the combination cottonseed meal and alfalfa
hay, which showed a slight effect.

No symptoms of toxicity were noted as a result of feeding
rations containing cottonseed meal over a period of seven weeks.

When suitable rations are provided, the albino rat consumes
an enormous amount of feed’in proportion to its live weight.

The writer desires to express his appreciation of the assist-
ance of Dr. H. H. Mitchell in outlining the method used in
this investigation and for many helpful suggestions. He is also
indebted to Dr. H. 8. Grindley for his encouragement and gen-
eral supervision of the thesis problem.

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THE JOURNAL OF DAIRY SCIDNCE, VOL. Iv, NO. 6

588 W. B. NEVENS

REFERENCES

(1) Ricuarpson, A. E., anp Green, H.S8.: Jour. Biol. Chem., 1916, xxv, 307;
ibid., 1917, xxx, 243; ibid., 1917, xxxi, 379.
(2) Menvet, L. B.: Jour. Amer. Med. Assoc., 1915, Ixiv, 1539.
(3) Osporne, T. B., anD MrenveL, L. B.: Zeit. physiol. Chem., 1912, Ixxx, 307.
(4) Osporne, T. B., anp Mrenpe., L. B.: Jour. Biol. Chem., 1916, xxvi, 293.
(5) OsBornz, T. B., anD MenveEt, L. B.: Jour. Biol. Chem., 1917, xxix, 69;
ibid, 1917, xxix, 289; Proc. Soc. Exp. Biol. and Med., 1915-16, xiii, 147.
(6) McCottum, E. V., AND Simmonps, N.: Jour. Biol. Chem., 1917, xxxii, 347.
(7) Hart, E. B., anp Humpurey, G. C.: Jour. Biol. Chem., 1917, xxxi, 445.
(8) Hart, E. B., anp Humpurey, G. C.: Jour. Biol. Chem., 1918, xxxv, 367.
(9) Fraps, G. S.: Bul. 128, Texas Agr. Exp. Sta.
(10) Henry, W.A., anp Mornison, F.B.: Feeds and Feeding. 15thed., Appen-
dix table 2.
(11) Menpext, L. B., anp Finn, M.8.: Jour. Biol. Chem., 1911, xi, 1.
(12) Raruer, J. B.: Bul. 163, 19138, Texas Agr. Exp. Sta.; Jour. Amer. Chem.
Soc., 1914, xxxvi, 584.
(13) Pomasx1, A.: Soobshch. Biuro Chastn. Rast. (Petrograd), 1915, II, no. 2,
3-12; Exp. Sta. Rec., xxxvi, 805.
(14) Wetts, C. A., anp Ewina, P. V.: Bul. 119, 1916, Ga. Agr. Exp. Sta.
(15) Editorial. Exp. Sta. Rec. 1910, xxii, 501.
(16) Dinwippiz, R. R.: Bul. 76, 1902, Ark. Agr. Exp. Sta.
(17) WitHEeRs, W. A., AND Brewster, J. F.: Jour. Biol. Chem., 1913, xv, 161.
(18) Wiruers, W.A., anp Carruta, F. H.: Jour. Agr. Res., 1915, v, 261; Science,
1915, xli, 324; Jour. Agr. Res., 1918, xii, 83; Jour. Biol. Chem., 1917,
Xxxil, 245.
(19) AuspErRa, C. L., anD Scuwartz, E. W.: Jour. Pharm. and Exp. Therap.,
1921, xvii, 344.
(20) MircnEet, H. H.: The Nutritive Value of the Protein Mixtures of Food-
stuffs at Different Levels of Intake. Unpublished manuscript.
(21) OsBorne, T. B., anpD MrenpeL, L. B.: Jour. Biol. Chem., 1917, xxxii, 309.
(22) OsBornzu, T. B., anD Wakeman, A. J.: Jour. Biol. Chem., 1919, xl, 383.
(23) Waters, H. J.: Proc. Soc. Prom. Agr. Sci., 1908, xxix, 3.
(24) Aron: Biochem. Zeit., 1910, xxx, 207.
(25) Fourin, O.: Amer. Jour. Physiol., 1905, xiii, 66; ibid, 1905, xiii, 117.
(26) LanpeRGREN, H.: Skan. Arch. fur Physiologie, 1903, xiv, 112. -
(27) Catucart, E. P.: Biochem. Zeit., 1907, vi, 109.
(28) Tuomas, Karu: Arch. Anat. und Physiol., Physiol. Abt., 1919, 219.
(29) Rusnur, M.: Arch. f. Hygiene, 1908, Ixvi, 1.
(80) Micuaup, L.: Zeit. physiol. Chem., 1909, lix, 405.
(81) Catucart, . P.: The Physiology of Protein Metabolism. 1912, p. 68.
(82) SHerMan, H. C.: Jour. Biol. Chem., 1920, xli, 97.
(83) Lewis, H. B., Dunn, M.S8., anp Dorsy, E. A.: Jour. Biol. Chem., 1918,
XXXvi, 9.
(84) Mircuety, H. H.: Jour. Biol. Chem., 1918, xxxvi, 501.
(85) OsBporNneE, T. B., anp Mrenprt, L. B.: Jour. Biol. Chem., 1914, xvii, 325.

BIOGRAPHY

The writer was born at Winnebago, Illinois, November 27,
1885. Academic training: graduated from Rockford High School
in 1905; attended Wheaton College 1905-6 and College of Agricul-
ture, University of Wisconsin, 1911-14; Graduate student, Univer-
sity of Illinois, 1914-17 and 1919-21. Degrees: B.S., University of
Wisconsin, 1914; M. S., University of Illinois, 1917. Positions:
Assistant in Dairy Husbandry, University of Illinois, 1914-17:
Assistant Professor of Dairy Husbandry, University of Nebraska,
1917-19. Honor Societies: Alpha Zeta, Gamma Sigma Delta, Phi
Lambda Upsilon and Sigma Xi.

PUBLICATIONS

1. Raising Dairy Calves. Ext. Bul. 51, Nebr. Agr. Exp. Sta., Aug., 1918.

2. Some Factors Affecting the Cost of Milk Production. Ext. Bul. 55, Nebr.
Agr. Exp. Sta., June, 1919.

3. The Arrangement of Rectangular Dairy Barns, Cir. 199, Ill. Agr. Exp.
Sta., June, 1917. (Jointly with R. S. Hulce).

4, Feed and Care of the Dairy Calf. Cir. 202, Ill. Agr. Exp. Sta., Aug., 1917,
(Jointly with R. S. Hulce).

5. Care and Management of the Dairy Herd. Cir. 204, Ill. Agr. Exp. Sta.,
June, 1919. (Jointly with R. S. Hulce).

6. Purebred Sires Effect Herd Improvement. Cir. 8, Nebr. Agr. Exp. Sta.,
July, 1919. (Jointly with M. N. Lawritson and J. W. Hendrickson).

7. Dairy Barn and Milk House Arrangement. Cir. 6, Nebr. Agr. Exp. Sta.,
Oct., 1919. (Jointly with J. H. Frandsen).

8. Breed and Size of Cows as Factors Affecting the Economy of Milk Pro-
duction. Jour. Dairy Sci. Vol. II, No. 2, Mar., 1919.

9. The Preparation of a Dairy Exhibit. Journal Dairy Sci., Vol. II, No. 5,
Sept., 1919.

10. The Self Feeder for Dairy Calves. Jour. Dairy Sci., Vol. II, No. 6, Nov.,
1919.

11. The Quantitative Determination of Amino-Acids of Feeds. Jour. Biol.
Chem. 1921, XLVIII, 249-272. (Jointly with T. S. Hamilton and H. S. Grindley).

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