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REPORT OF TI=[E COMMITTEE ON METHODS OF
INVESTIGATION
]~ETHODS OF E X P E R I M E N T A T I O N IN ANIMAL NUTRITION
Preparation of Plans
Prepare clear, accurate, comprehensive, and detailed plans of the
specific investigation which has been selected. The plans, in a tentative form, should be submitted in full, for criticism, to the members of the department, and to the Director of the Station. No investigation should be started until the plans for the same have been
thoroughly matured and perfected. Each phase of the work of the
investigation as outlined below should be ~ v e n careful and thorouch consideration and study in the preparation of the plans.
Study of the Literature
The first essential in properly outlining an investigation is a thorough knowledge of the previous investigations on the specific subject, and also on the closely related subjects. Such knowledge can
be gained only by a thorough study of the literature. In this study
the preparation of abstracts of methods and results, and the compilation of a bibliography are helpful.
Equipment for Digestion and Metabolism Experiments
(a) For swine. The metabolism crafts described and used by McCollum and Steeubock of the Wisconsin Agricultural Experiment
Station (Research Bul. 21, 80-96, 1912) and by Forbes of the Ohio
Agricultural Experiment Station (Circular 152, 75-85, 1915) meet
the requirements fo~ either digestion or metabolism experiments:
(b) For sheep. The metabolism crates described by MeCollum
and Steenbock and by Forbes for s~vine can equally well be used for
sheep, with slight modifications, especially as to the size of the mesh
of the wire screens used for the floor, and the shape and the size of
the feed boxes. The metabolism cage described by Gies (Amer.
Journ. of Physiology 14, 403-412, 1905) could also be used to advantage with sheep.
(c) For cattle. The digestion and metabolism stalls described and
used by Armsby (Pa. Agr. Exp. Sta. Bul. 42, 74, 1898) and by Mumford, Grindley, Hall, and Emmett (Ill. Agr. Exp. Sta. Bul. 172, 237,
1914) meet the requirements for either digestion or metabolism experiments with cattle.
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(d) F o r horses. The digestion and metabolism stalls described
above for cattle could beused, with minor modifications, for digestion and metabolism experiments with horses.
With both cattle and horses digestion and metabolism investigations may be conducted without metabolism stalls by having competent attendants catch the excreta in weighed receptacles.
Selection of Experimental Animals
(a) The species of animal selected will depend to a considerable
degree on the nature of the investigation. For digestion experiments any of the different species of farm animals may be used. For
metabolism experiments the species to be used should be determined
with much care, considering the rapidity of growth, reproduction,
or other increase, the vigor of the response to feed, the adaptability
to the necessary restrictions of the experimental routine, and the
like.
Because of their voracity, rapid g r o w t h and prolificacy swine
have been found well adapted for metabolism investigations. Among
the ruminants sheep also readily lend themselves to successful metabolism experiments.
Certain lines of animal nutrition investigations, for example, feeding experiments with isolated proteins and with isolated amino
acids, can be carried on to advantage with small animals such as
mice, rats, and rabbits, as well as with the smaller farm animals
such as pigs a n d sheep. In deciding on t.he species of animal to use
it must be borne in mind that experiments with the larger animals
involve much larger expenditures of time, labor, and food materials
than do experiments with small animals. It should also be considered, in this connection, that while the fundamental laws of growth
are in all probability quite similar, if not identical, for farm animals
a n d for small laboratory animals, our practical scientific interests
are not confined to those fundamentals in which all vertebrate animals agree, but are based largely upon physiological superficials,
upon differences in emphasis or adcentuation of the same set of fundamental principles. The economic usefulness of results of nutrition investigations is not necessarily assured by the establishment
of the fundamental principles involved, but is apt to require a demonstration of the facts of interest as applying to the species of interest. It is well, therefore, in the choice of the species of animal to
be used in a nutrition investigation, to bear in mind the practical
advantage of using as subjects, wherever it is practicable, the species to which the results are to be applied. In this connection the
experimenter should also have in mind the ~udience whom he would
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reach with his results. The agricultural public is more easily and
effectively reached with results from experiments with farm animals
than with results from investigations with other species which, because of considerations of economy of time, labor and money, are
more commonly used for laboratory studies in the field of nutrition.
(b) Breed and type of ~n~m~. In digestion and metabolism experiments all of the subjects should be of the same type and breed.
It is also desirable that all animals used in the same experiment
should have been produced in the same herd or flock, by the same
sire or from closely related sires, and from dams which are as nearly
as possible uniform in age, breeding, and type. A definite and accurate description of each animal should be recorded at the beginning of the experiment.
(o) Sex. It is advisable that animals used in digestion and in
metabolism experiments, except those involving milk production or
other female functions, should be castrated males, because of the
greater facility with which the excreta may be collected without
contamination.
(d) Age. All animals in the same experiment should be practically of the same age; and the exact age of each animal should be
known.
(e) Previous treatment. The experimental animals should preferably be selected from the salae herd or flock, identical previous
treatment of all subjects being highly desirable. If it is necessary
9 to select animals from different herds, or flocks, or from different
localities, they must be submitted to a preliminary feeding period
of considerable length (depending on the nature of the investigation) during which all must be fed alike. 0nly animals which are
apparently healthy and normal, and apparently functionally alike,
should be selected for nutrition experiments; and any such tests as
are effective to prove them so should be made.
(f) Number of animalB. In digestion and metabolism experiments
not less than three animals should be used in each lot. If practicable
five or even more should be used. Results obtained with four, five,
six or more animals are much more reliable than those obtained with
one or two, because of the reduction of the influence of individuality.
Selection and Sampling of Feeds
(a) Selection. The feeds selected for nutrition experiments
should be of the best possible grade. Before the beginning of the
experiment, a sufficient quantity of each of the feeds should be selected, from the same source if possible.
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(b) Sampling. The feeds used in digestion and metabolism experiments must be thoroughly mixed so that each portion fed will be of
the same composition. Thorough mixing is also necessary in order
that representative samples can be obtained for chemical analysis.
Whenever foods are weighed out for nutrition investigation samples must be taken for moisture estimation. On this account it is
desirable that all portions of feed which are to be administered during an experimental period should be weighed at one time. In other
words, the feeds or the rations must not vary in chemical composition during the experiment in which they are used, and the samples
of the feeds taken for chemical analysis must represent exactly the
foods as they are weighed out for feeding.
The following is an approved method of sampling grains either
whole or ground: The amount necessary for one experimental period
is spread out to a thickness of six to twelve inches on a clean and
perfectly tight floor. Ib is then shoveled systematically by two men
standing on opposite sides of it who throw alternate shovelfuls of
the grain into one pile. The grain is then spread out again, divided
into quarters, mixed from opposite sides and the two resulting pries
combined into one. This last procedure is repeated. The grain is
then spread out into three approximately equal lots. The weighing
of the individual feeds for each of the animals for an exPerimental
period is begun immediately after it has been thoroughly mixed as
described above. Special care should be taken in the weighing to
have the individual feeds as nearly uniform in character as possible.
With this in view, a part of each feed may be taken from each of
the three piles. The individual feeds may be kept in bags of paper
or cloth, or in eovered pails, properly labeled to designate the animal for which the feeds were weighed. After the weighing of every
two or three portions a small amount of the thoroughly mixed grain
may be taken from each of the three piles to form a composite sample for chemical analysis.
Hays, silage, stover, etc., must be cut into lengths of one inch or
less, since it is impossible to prepare samples of these feeds before
cutting them, which are aeeurately enough representative of the
whole amount to be satisfactory in digestion and metabolism experinlents. The cut hay, stover, etc., may be sampled and weighed exactly as outlin'ed above for the grains. In the ease of silage the
individual feeds may be stored in cloth bags, in a refrigerated room.
No harm has been found to result from the freezing of the silage,
but it must be allowed to thaw before feeding. The bags containing
individual feeds of silage should not be piled, one on top of another,
for storage, before they are frozen. If one does not practice the
freezing of the silage it must be sampled for analysis each time it ia
weighed out for feeding:
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105"
(c) Water. In investigations involving mineral metabolism the
water given to the experimental subjects must be distilled, unless
the nature of the particular problem should render this unnecessary.
(d) Amount of feed. There should be no change in the daily
amount of feed given during an experimental period. It is, therefore, highly desirable that all feed given be consumed without waste.
Length of the Exper~ent
(a) Preliminary feeding. The experimental subjects should be
~ubjected to a preliminary feeding (7-10 days for horses or swine;
10-12 days for ruminants) to allow for the complete removal from
the alimentary tract of all residues from previous rations. During
this preliminary feeding the rations must be fed in tba same quantity as during the experimental period to follow.
(b) Collection period. In digestion experiments the collection period should be not less than ten days for swine and horses, and not
less than twelve to fifteen days for ruminants. In metabolism in~/estigations the preliminary and the.test periods should be at least as
long as in digestion experiments, and in most such studies should be
of greater duration.
(c) Mark-lng of feces. With human beings and swine an accurate
end-point in the collection of the feces may be reached by the feedi n g of an inert, indigestble substance which, by its color, in the
feces, will serve as a marker. The markers used are lamp black and
carmine. Of the two, carmine, because of its intense color, has the
wider usefulness. ]n the form of a powder it is fed mixed with the
food. In accordance with the color of the feces, as determined by
the nature of the diet, a n d with the size of the animal, 0.250-1.000
gin. of carmine will give a distinct color to the feces. With ruminants the accurate marking of the feces is not possible. In this case
take a given number of days' defecation.
Experiments to Determine the Nutritive Value of Feeds, Rations,
and Isolated Nutrients, Such as Proteins, Lipoids,
Animo Acids, Etc. ,
Nutrition experiments of short duration, even when accompanied
by elaborate chemical and physical analyses and examinations, give
but imperfect insight into the final nutritive value of a feed, ration,
or isolated nutrient, for long-continued normal and healthy growth.
It is essential, therefore, that experiments of this character be of
extended duration. Such experiments should cover at least one life
cycle, and in some cases several such periods in succession a r e
needed in order to exhibit the final effects of a dietetic regime.
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T H E A M E R I C A N SOCIETY OF A N I M A L PRODUCTION
The Care of the Animals
(a) Feeding and watering. In digestion and metabolism experiments the animals should be fed regularly from two to four times
per day, and in such manner as to prevent loss of feed. The animals
should also receive their water at regular times, and in unrestricted
amounts. The salt given should be weighed. It will be consumed
with least waste if it is fed mixed with the feed.
It is fatal to the accuracy and the reliability of digestion and metabolism experiments if the rations be not eaten regularly and in
uniform quantities. It is also essential in all investigations of normal metabolism, that the animals experimented upon shall remain
in all ways in that perfectly healthy state in which the results are
to apply.
(b) The weighing of the animals. Initial and final live weights
should be taken on at ieast three successive days. In many investigations the taking of daily weights i s desirable. All live weights
should be taken under as nearly uniform conditions as possible.
(c) Exercising of animals. When the exercise afforded by the
method of the experiment or the character of the metabolism crate
or stall is sufficient for the maintenance of normal functional activities the" animals must be given such additional exercise as they
require, at regular times and in definite ways.
(d) The cleaning of the crates or stalls and the animals. A rigorous daily cleaning of the crates, stalls and animals is necessary to
insure the health and comfort of the experimental subjects, and to
prevent the contamination of the excreta. The method of this
cleaning will naturalIy be determined by the conditions of the investigation.
The Collection end the Sampling of the Refused Feeds
At times, especially when the experimental animals are full-fed,
they will leave some of their feed. In some investigations the waste
from one feeding, either in its natural condition or after drying and
grinding will be consumed if mixed with the next feed, but judgment is needed in such practice to avoid throwing the animals off
feed. Ordinarily the refused feed will be preserved from day to
day, and at the end of the experimental period will be mixed and
sampled for analysis. It is highly desirable that there be no refused feeds, since the results will represent not the feed as it is
weighed, but only that portion which is eaten, which may differ
much iS composition from that which is left; but if there be refused
feed this must be collected, handled, and accounted for with most
scrupulous care.
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The Collection and the Sampling of the Feces
At the end of each experimental day, the f e c e s m u s t be Weighed,
mi~ed, and sampled for analysis. The feces of cattle and horses
can be satisfactorily mixed in a pressed-steel wheel-barrow by the
use of a mortar hoe. After thus mixing thoroughly, a definite aliquot portion of the dail 5, feces is taken to form a part of the composite sample for the experimental period. This aliquot is obtained
b y taking small portions at random from the entire mass of the ~horoughly mixed feces. The portions thus taken are transferred to
tared friction top cans which have been thymoled by rinsing with
a ten per cent alcoholic solution of this preservative, and then drying. When an aliquot portion of a day's feces has been transferred
to one of these thymoled cans, approximately five grams of finely
ground thymol is sprinkled over the feces. This should be thoroughly stirred into the entire sample with a proper sized trowel
which is cleaned by scraping it upon the sides of-the can. The can
containing the sample should be tightly closed, properly labeled,
and placed in a cold storage room maintained at the lowest attainable temperature.
At the end of the experimental period, the daily samples of the
feces are taken from cold storage and thoroughly mixed with hoe
and trowel. From the resulting composite sample representing the
feces for the entire experimental period smaller samples are taken
for analysis. Samples to be preserved by refrigeration should be
inclosed in thymoled friction top pails or other tight receptacles. If
refrigeration facilities are limited other samples should be taken in
shallow enamelled pans for preservation by drying. It is recommended that these samples be air-dried at 50-60 C. for one week,
and that they be stirred each day during this time. They should
then be exposed to the open air at the ordinary temperature for four
or five days, and should then be weighed, put into tight receptacles
and sealed with paraffin. They may well be kept in cold storage.
These air-dried samples are reserved for emergency use in case the
results of the chemical analysis of the fresh samples are not satisfactory ; or in case, after the analytical work upon the fresh samples
has' been completed, it seems desirable to undertake further investigations involving the analysis of the feces. In such cases, the
fresh samples of the feces may have undergone deterioration such
as would render them unfit for further study, the air-dried samples
remaining in better condition for the intended purpose.
In digestion and metabolism experiments with pigs and sheep the
feces may be sampled as described above for cattle and horses, or
since the daily feces are much less in quantity than with cattle and
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horses, the entire feces for the day may be placed without weighing
into tight, thymolized cans, for preservation in cold storage. At the
end of the experimental period these cans each containing one day's
defecation should be removed from cold storage and weighed. The
feces can then be mixed and sampled for preservation and analysis.
Sheep and swine feces may often be most satisfactorily prepared
for mixing and sampling by repeated grinding through a sausage
mill. Feces may be ground very readily when partially frozen.
The Collection and Sampling of the Urine
Urine may be quantitatively estimated, and sampled for preservation and analysis either by weight or by volume. In case the
urine is to be estimated by weight, the receptacles and other apparatus used in its collection, transfer, measurement, and sampling must
each be weighed before and after use, the difference represeutiag
adherent urine; or these small amounts of urine may be recovered
by cleaning with water, and may be accounted for by separate
analysis. To follow this method with particularity requires much
weighing; and the accurate weighing of large containers and large
volumes of liquids presents some practical difficulties. In practice
it is customary either to compensate for certain of these small
amounts of urine adhering to apparatus by the use of arbitrary corrections, or to ignore these amounts as inconsiderable. If the urine
as collected from the metabolism apparatus is estimated by weight,
the aliquot samples preserved and the portions used for each analytical determination must also be estimated by weight; special
facilities often being required for the accurate weighing of the
large volumes of urine needed for some estimations; or if measured
by volume a specific gravity determination will be required in the
computation of results. The advantage of estimating the urine by
weight is that its cancentration remains normal, a condition which
is essential in certain kinds of investigation, but of no importance in
all those investigations in which the data sought are merely total
.quantities of matter other than water in the urine. The disadvantages of this method are that it requires much labor and that this
fact inclines the experimenter either to ignore or to estimate by arbitrary correction certain small portions of urine adhering to apparatus which by other methods are conveniently and accurately accounted for. A further complication is introduced and a correction is required by the necessity, in the sampling of some urines, of
adding solvents to get into solution various solids which have separated out.
In estimating urine by-volume all receptacles and other apparatus
used in its collection and transfer may be cleaned with distilled
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water, the cleaning-water being added to the main portion of the
urine before its estimation. The combined urine and wash water
may then be made up to volume in graduated flasks; aliquots for
preservation and analysis may be taken by volume, and individual
portions for the chemical determination may be. measured by voli~me, either directly from the sample or after an intermediate measurement and dilution in a graduated flask. The advantages of this
method over the preceding one, for most purposes, are very great.
Maximum accuracy is attained at minimum expenditure of labor,
and no difficulty is introduced by the necessity of adding solvents to
the urine in sampling. The disadvantages of this method are that
the normal concentration of the urine is lost, and that the integrity
of the samples depends on the purity of the distilled water.
For some purposes it is desirable, as previously suggested, to make
separate samples and analyses of the main portion of the urine and
of the cleaning water.
In metabolism experiments with pigs and sheep, using the metabolism crates of either McCollum and Steenbock, Forbes, or Gies, the
urine should be collected in thylmoled, glass bottles.
In metabolism experiments with, steers and horses using the
Armsby or the Mumford, Grindley, Hall, and Emmett metabolism
stalls, the urine should be collected by the Armsby rubber funnel in
thymoled, enameledpails which are provided with lids having twoinch circular holes in the center, to admit the end of the rubber funnel. The pails should be placed under the stalls in enameled dish
pans which will serve as catch traps in case of accident.
In metabolism experiments in which excreta are caught by attendants, tin, galvanized or enameled pails may be used in the collection. The urine will then be transferred to thymoled glass bottles. In experiments with cows, scoop shovels are useful in collecting excreta passed while the animals are lying down, as also are
emergency pans in the floor or in the gutter behind the cattle.
The urines collected as above may be taken to the refrigerator at
regular intervals during the day, or only at the end o f the experimental day, as determined by the weather, the character of the
urines, and the conditions of the investigation. The used containers
are exchanged in each case for clean ones, which are likewise coated
with thymol. At the end of the experimental day the urines are
mixed and sampled by some such procedure as one of those previously outlined. Certain samples may be used for daily chemical
or physical determinations; other daily aliquots will be retained in
the cold storage room until the end of the experimental period. During this time it is desirable that the urines should not be frozen, as
this throws down solids which it may be difficult again to get into
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TIIE AMERICAN SOCIETY OF ANIMAL PRODUCTION
solution. The temperature, therefore, should be as close as practicable to the freezing point.
At the end of the experimental period the daily aliquot samples
of urine are r e m o v e d from the cold storage, and are combined,
mixed and sampled for further preservation and for analysis. In
the preservation of the composite samples a temperature just above
freezing will be satisfactory for many purposes; for others the lowest attainable temperature may be inadequate. The requirements
for the preservation of urine for specific purposes should be determined by actual trial.
Hair, Scurf, and Sk-iu Secretions
In metabolism investigations of the greatest accuracy, involving
the complete chemical accounting for the ration and for the growth
of the animal, shed hair and scurf, and ski.n secretions removed by
brushing or scrubbhlg must be collected and saved in a systematic
manner, and subjected to chemical analysis. Inasmuch as this material will be partially in the nature of an exeretum and partially
accreditable to the body of the animal, measures may be taken for
the separation of thege fractions. This matter is not significant in
relation to digestion experiments.
The Methods for the Chemical Analysis of the Feeds, Feces and
Urine
The methods of the Official Agricultural Chemists of the United
States as given in the revised edition for 1908 of Bul. 107, of the
Bureau of Chemistry U. S. Dept. of Agr., should be used in the a n alysis of the feeds, the feces, and the urines.
The Recording, Calculation and Tabulation of the Experimental
Data
(a) The recording of the data. All data and observations should
be carefully, fully, and legibly recorded, in a systematic manner, at
the time they are made, in proper record forms of one kind or another. Such original experimental records should be kept in a fire~
proof vault, or if this is not possible, duplicate copies should be
made, each day, and the copies or the original records deposited
in the vault. In so far as possible printed blank forms should be
used in recording the data.
(b) The calculation and tabulation of the data. The experimental
data should be posted each day on proper record sheets or in proper
note books; and the calculation and tabulation of the results should
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be carried forward each d a y as far as the data permit so that the
results may be of use in directing the course of the experimental
work from day to day, and week to week.
The Interpretation of the Results
The interpretation of the results of nutrition experiments is a
subject in itself which should receive the independent consideration
of the committee.
The Committee on Methods of Investigations are much indebted
to Dr. E. B. Forbes for valuable suggestions and assistance in the
preparation of this report.
(Signed) H . S . GRINDLEY,Chairman,
W. A. Cocn~-~,
J. M. EVVARD,
L. D. l=[AI2~,
W. A. CAnRO~a~,
Committee.