A STUDY OF THE TOXICITY 0F
GOSSYPOL

THESIS FOR THE DEGREE OF M. 8.

Donald E. Wilcox
1931

 

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A STUDY OF THE TOXICITY

0F GOSSYPOL

A. STUDY OF THE TOXICITY 0F GOSSYPOL

Thesis

Respectfully submitted to the Graduate School
of Michigan State College of Agriculture and
Q

Applied Science in partial fulfillment of the

requirements for the degree of Master of Science.

By

Donald E. Wilcox
N...

1931

 

IHE‘IS'

acmom GENTS

The author of this thesis wishes to acknowledge his appreciation
of the assistance given by Professor 0. F. Huffman.and mr. L. As Moore
of the Dairy Experiment Station, in planning and conducting the eXperi-
ment and in the preparation of this manuscript.

He also wishes to acknowledge his indebtedness to Professor E. L.
Anthony, Head of the Dairy Dapartment, for his kindly criticism in the
preparation of this manuscript.

The author is also indebted to Dr. C..A. Happert, Associate Pro-
fessor of Chemistry, for his assistance in the planning and conducting
of the experiments with rats.

He also wishes to express his appreciation.to Dr. C. S. Robinson,
Experiment Station Chemist, for assistance given.in.making the gossypol

analysis.

TABLE OF CO:TEHTS

I Introduction

II Review of Literature

1. Manufacture of Cottonseed Meal

B Feeding Experiments with Special Reference to Cotton-
seed Lieal Injury

1.
2.
5.
4.
5.
6.

7.

Experiments with Cattle

Eneriments with Sheep

Experiments with Horses and Rules

Experiments with Swine

Experiments with Pigs Following Steers Receiving
Cottonseed Meal

Experiments with Poultry

Experiments with Hats

0 Theories of the Cause of Cottonseed Heal Injury

1.

2.

5.
4.
5.
6.

7.

Choline and Betaine

Parasitic Organisms and Decomposition Products of
Bacterial Action

High Protein Content
Phosphoric Acid
Acidosis
Toxic Principle in the Protein molecule
Deficiency Disease Theories
a Vitamin B
b Iron Deficiency

0 Factor or Factors Carried by Hay

page

12
15
16

21

58

i3

26

27

28

29

29

31

page
8. Gossypol 57
D The Chemistry of Gossypol 45

E Formation and Occurrence of Gossypol in the Cotton Seed 48

F The Effect of Various Supplements in Counteracting the 56
Injurious Effects of Toxic Substances

G Discussion of Review of Literature 58
III Experimental Work 50
A Object 60
B Original Plan of Experiment with Dairy Heifers 60
1. Procedure 60

a ' Animals Used 60

(1) Age 60

(2) Health 61

b Choice of Rations 61

(1) Milk 61

(2) Grain 61

(3) Roushase 61

(4.) Minerals ' 61

c Management 62

(1) Water 62

(2) Shelter 62

(3) Sunlight 62

(4) Care 62

(5} Feeding Methods 63

2.. Collection of Data 63

a Feed Records

(1)
(2)
(5)
(4)

(5)
(6)
I7)

b Reproduction

(l)
(2)
(3}
(4i
(5)
(6}

0 Observations

(1)
(2}
(5)
(4)
(5)
(6)
(7)
(8)
(9)

P869

63

Feed Consumed 63
nutrients Required 63
Nutrients Consumed 63
Per cent of Raw Cottonseed meats in the 63

Ration
Amount of Gossypol Ingested 63
Per cent of Gossypol in the Ration 64
Rfilligrams of Gossypol per Kilo of Body 64
Weight

64

Oestrual Periods 64
Breeding Data 64
Ease of Parturition 64
Placenta 64
Recovery 64
OffsPring 65
65

Weight 65
Height at Withers 65
Consistency of Feces 65
Health 65
Appetite 65
Blindness 66
Stiffness and Abnormalities in Gait 66
Oxygen Carrying Capacity of the Blood 66
Photographs 66

page
C Procedure _ 66
1. Animals Deed , 66
2. Choice of Rations 66
5. Management 67
D Data
1. Feed Record 67
2. Reproduction 69
3. Observations 69
E Experiments with Eats 71
1. Animals Used 4 71
a Age 72
b Weight ' 72
c Sex 72
2. Nanagement 72
a Cages 72
b method of Feeding 72
0 water 72
3. Diets Used 72
a Series I 72
b Series II 74
0 Series III 75
4. Collection of Data 78
a weight 78
b Growth 78

0 Health 78

 

 

P886
F Determination ofGossypol in Samples of Cottonseed meal 78
and Raw Crushed Cottonseed.Meats

»1. Collection of Samples 78

2. Esthod of Analysis 78

3. Results 79

G Discussion of Experimental Results 80

H Summary 85

IV Literature Cited 86
7' Appendix. 103
A Tables 104

B Graphs 119

C Plates 133

INTRODUCTION

Economical milk production in regions where alfalfa hay is not
readily grown depends to a large extent upon the cost of protein con-
centrates needed to balance the ration. In.E1chigan, although alfalfa
is increasing in favor, much of the hay that is fed is low in protein,
and lack of sufficient protein.in.the ration is very often a limiting
factor in milk production.

Ordinarily cottonseed meal is the cheapest high protein concenp
trate that can be purchased, and the biological value of its protein
has been shown.by feeding experiments to be relatively high. Its use
as a cattle feed has been somewhat limited, however, because its use
in fairly large amounts is thought to produce injury. Several experi-
ment stations and some of our leading texts on cattle feeding recommend
the use of cottonseed meal only in limited amounts.

Cottonseed meal injury has been attributed to the presence in it
of chloline, betaine, pyro-phosphoric acid, bacteria and molds, protein
decomposition products and gossypol.

The injury has also been attributed to deficiencies in.the rations
of which cottonseed meal is a part such as, a lack of vitamin.B, vita-
nun.A, iron, calcium, and a factor or factors carried by good quality
hay.

Gossypol has been found to be very toxic where fed to some species
of animals. There is no direct evidence, however, to show whether or
not it is toxic to dairy cattle when fed in fairly large amounts with a

ration that is otherwise adequate.

“7

*v

 

a. purpose a: this investigation was to Italy the toxicity of
panel te growing dairy heifers, when fed, as it naturally eocurs,
in raw cottonseed kernels. i ration which supplies all of the known
factors essential for growth, nintenance and health was fed in con-
Janction with the re. cettenseed. in attempt was also Ids, using rats,
to determine some facts regarding the physiological effects of feeding
cessypol as they are affected by various additions to an otherwise normal
ratien. a may of the «mace ef cossypol a ran cottonseed an! in
cettonseed meal is else included. i

BENIN OF LITERATURE

the use of cottonseed and eottoneeed products as food dates back
approxintely one humired years. the Royal Society of Arts of Iondon
(l) in 1785 offered nodals'to the person who would patent a process for
expressing the oil froa the seed of cotton and non. of the residing
seed, hard and dry cakes as food for cattle. he offer required that
one ton of oil should be expressed and 500 pounds of dried cans be ob-
tained to win the gold nodal. hr the preparation of one half as nah
nterial a silver nodal was offered. Probehly the large quantities re-
quired defeated the purpose for although the pro-dun were offered fer
six years no process was presented.

on. South carolim laicultural Society to) in 1705 offered a pre-
niun for the patent of a process fer the extraction of oil tron cotton-
seed. Eewever, it was not until 1910 that Daniel dillet of Springfield,
fissacluisetts (2) patented a process for preparing food from cottonseed.
m 2.11.135... a proceeo m- attracting oil fron the seed was pat.
entod by George P. Disses of Virginia.

lo doubt cottonseed was used as a food for cattle to a limited ex-
tent before cottonseed noel was placed on the nrhet. It was not until
about 1850, however, that cottonseed products boos-o courcially inert-
aut as food products. Prior to the civil Iar there were but seven cotton.-
.1... .111- m the south. Since that the the growth of the eottonseed
oil and cottonseed and industry has been very rapid. cottonseed, fora-
erly almet wholly wasted, now is node up into products having a yearly
value estinated at $250,000,000 (8). dpproxintely one-third of this

o
.
t,—

I.

amount is obtained from cottonseed oil meal and cottonseed cake.
manufacture of Cottonseed Meal

The process of manufacture of cottonseed meal may have a marked
influence on the presence of gossypol in the finished product due to the
effect of heat and pressure in converting the "free gossypol to a prod-
uct known as ”bound gossypol" or "D-gossypol" a substance much less
toxic than.the original ferma Fer this reason a short description of
the process of cottonseed meal manufacture is included here.

The cottonseed on.reaching the oil mills is first screened to remove
sand, dirt, bolls, and foreign.substances, and finally a draft of air is
used to complete the cleaning process. The seed is now ready for the
linters. These machines are an elaboration of the ordinary cottonpgin;
and whatever staple remains upon the seed is stripped off in passing
through.them. From.the linters the seed passes to the huller, a high
speed cutting machine which cuts it up thoroughly. The hulls, by screens
and heaters are now separated from.the meats, which latter are, by scrsw
conveyors, conducted to bins contiguous to roller crushers, and as fast
as required are passed through the crushers where the mass is reduced to
a uniform.consistency, and is known to millmen.as "uncooked meal".

In the mnnfacture of cottonseed meal from this raw product the raw
kernels are usually cooked before the oil is expressed, to increase the
yield of oil. The details of the cooking vary to a considerable extent
in.different mills.

A few mills use the "expeller", a cold-press method in which the
decorticated raw kernels are usually dried in.a pro-heater and are then

passed into the expeller preper. In the eXpeller they are forced by

pressure through cull orifices, this operation subjecting then to a
pinding action which squeeses out the oil. Both the dry heating ad
the grinding action are inortant in connection with conversion of ges-
sypol to bound gossypol or D-gossypol in the cottonseed noel.

so. .r‘ the mills use to. cpen kettle process in the manor.
of the meal. In this nethod the deccrticated cottonseed hernels are
agitated in large eteen-Jacheted bottles until they are are or less
thoroughly co‘inuted. lhe tins of cooking and the stoen pressure (to-p-
erature) in the Jacket vary greatly. After cooking the kernels pass to
a cake fer-or and then to a hydraulic press where the oil is expressed.
no press cake is thenground to a fine Ital.

' The continuous-cooker which is new used by a mot-1t: of nills,
eonsists of three to five steal Jacketed m... or stacks placed one above
the other. The raw hernels enter the upper stack an). proceed downward

as those below are emptied. There is considerable variation in the tin
required for the hernels to pass through the stacks am also the steel
pressure used in the stacks at the various nills. In either of the latter
nethods water or steam ny be added or a current of air passed through the
cocher in order to regulate the moisture content of the kernels.

The variation in nethcd of nnufacture of cottonseed meal as well as
variations in teqerature, pressure and noisture content within one process
as used by different nills nay have a nrked influence on the geesypol eon-
tent or the condition of th gossypol in the finished cottonseed Dal.

leeding Rxperinonts with Special Reference to Cottonseed lbal Injury

lunerous investigations have been conducted in the past fifty years
dealing with the use of cottonseed products as a food. my havo inclded
a study of the biological value of its protein, its replace-eat value in
the ration as conpared with other foods, its effect on the unlity and
quantity of butterfat produced when it has been fed to dairy cows, d
also a stub of their injurious effect when fed in large onounts.

Only those investigations having a direct bearing on cottonseed Dal
injury will be reported.

Yeelher (d) seems to have given the first published report of the
injurious .rroot. which occurred fron feeding cottonseed neal. In ion
he reported cases of injuryte sheep uni cattle inhglandwhichwas at-
tributed to the use of cottonseed noel. Other cases were reported in
Europe and in England a f. years later, but it was not until about 1890
tint the problem cane unler investigation in this country.
grunts 323.119.1522. Stone (5) in 188! reported that the practice
of feeding cottonseed hulls seens to have begun as early as 1070, soon
after the introduction of the oil industry. needing hulls end cottonseed
noel as an exclusive diet for cattle was started about 13.5. no harmful
effects fron feeding 25 to 50 pounds of hulls and five to eight penile of
eettonuednoaldailyhedbeenrepcrtedup to the tine thereportwas
published.

wright (6) fed £000 steers and heifers weighing frcn 050 to ”0 ponnlo
for aperiodflef three nonths (eons of then longer) on a ration ecnposed of
cottonseed hulls all! cottonseed Isl. light pounds of anal and 85 pounds

of hulls were fed per day. Bay was fed once a week and oftener if scour-
ing developed. The average gain in weight was about 75 pounds per nnth.
About one per cent of the cattle scoured and three of the auinls died.
Death was attributed to pleuro pnemoaia.

Lloyd (7) fed six lots of 10 cows each for 12 weeks (ll weeks on ex-
mifint) on rations in which cottonseeds, cottonseed neal, and corn were
fed inecnjunctionwithher-slahayandninedhey. no coweusedwere
”natives" and grade Jerseys. his food eaten and the nilk produced is
sunrisod below:

not I. hot II. Int III. hot 11‘. Int T. Lot 11.
Iowa lay lined m lined m fixed Hay

cottonseed Bu w hey hey corn
Go go 0.8.1. 0.3.1. o.”

lbs. lbs. lbs. lbs. lbs. lbs.
Bey 9.4 9.5 14.2 18.0 13.8 9.!
Cotton Sccd 9.6 9.9 - - - ..
0.8. Heal - 9.7 9.9
corn 9.9 9.75
Iilk Produced 8.3 7.9 10.9 9.9 8.8 9.8

rho nilk production 1. at, low in .11 cases but 1. probably in
part due to lack of producing ability on the part of the native cows.
During the 12 weeks that the cows were fed on these raticns n iniurisas
results were noted. _

aha-berlain (8) fed four steers for an 94 day feeding period on a
ration of cottonseed hulls, "ed libitun" (15-20 lbs. per m) and cotton-
ntni :4 pounds per deg. it"tho eni of the trial they were still m-
ing and appeared healthy but there were sons indications that the di-
gestion of the aninls had been iqaired.

i'ietse (9) in 1898 reported investigating several cases in which
the death ef‘calves was attributed to the feeding of cottonseed no.1.
The calves received three heaping liters per dog of cottonseed noel in
addition to skin nilk, hay, and linseed cake.

hory (10) one year later reported an investigation in which three
calves were'fed skin milk to which one-fourth to one-half pound of cot-
tonseednealwasadded. methroe calvesdied infronfeurto sixwoeks.
The death of two of the calves was attributed to a toxic principle in
the cottonseed noel.

Gautier all]. Larsen (11) in 1895 reported a nuber of cases of pois-
oning of calves which were directly traceable to the eating of cotton-
seed Dal.

hry and Iilgore (12) fed four oxen on a ration ccnpoeed entirely
of cottonseed no.1 and cottonseed hunt. Large want. of both cotton-
seed neal and cottonseed hulls were consuned. it the end of 92 and 185
days of feeding there was no inJury noticeable. Fairly good nine were
ndeasieshownintheou—rybelow.

cottonseed llcal Cottonseed m1. Period; “in in flight

Io. of Oonsuned Consulted days pounds
per do: per the
l 9.8 17.2 92 150
2 18.0 16.6 92 155
8 9.6 14.1 135 185
d 8.5 12.4: 155 240

Gonnell and Kyle (18) of the Texas Station reported in 1899 that
steers which were no 20 pounds of cottonseed lmlls and 4.5 poutnle of
cottonseed neal for a period of 100 days nde fair gains and showed no

injurious effects. They reported also the results of a large nuabor of

farmers and feeders on the use of cotton seed and cottonseed hulls. no

injurious effects were. reported but the majority of feeders fed the cot-
ton seed only for short periods of ties.

not (14.) in 1905 reported observing peculiar toxic sylptene in
draft onenwhich were receiving two pounds of cottonseed neal per day.

Be describe'etths condition as follows:

'hdenteus swelling appeared at the extrenitiee, the appetite being
undisturbed. In later stoges weakness of the hint quarters appeared and
in single uni-do disturbances of equilibriu. tour of 15 affected oxen
showed disturbances of vision. dppmntly they becene ecqletely blind
as was evidemod by their groping gait, and by their colliding with other
aninls and surrounding objects. Inc-intion of tin eyes revealed no
special lesions except nrked protrusion of the eye ball and abnornal en-
largenent of the pupil. he nest severely affected aninls were slaugh-
tered, and the remainder recovered after administration of laxatives ad
alteration of the diet including discontinue“ of the use of cottonseed
noel."

iichsls and Burgess (15) after reviewing the literature bearing on
cottonseed lee-l feeding concluded that when the health of cows is un-
favorably influenced it is in all probability due to one of the follow-
ifl causes:

(1) leading the nal in conjunction with unsuitable roughage; (I)
feedings-it ~in a stale or msty condition; and (s) feeding it in excessive
quantity. They believed tint derangenonts of the aninls' system results
' tron feeding cottonseed meal with cottonseed m1. where the latter for-s
an exclusive roughage. Such a ration they point out is unbalanced, con-

10
toiling In euess of nitrogenous utter, on this eu-sidedneee eelbilld
with the wood: indigestible homo of the mu is responsible for tho
ill health that soaetinss follows its use. they observed so ill offsets
tro- feodiug cottonseed stool as one-half or three-fourths of the grain
lixturewhsnthe Wanna earner sorchnnsilsce. live to six
pounds per day of sottonsesd neal was sou-stiles fed.

m (16) noted the unthritjzy audition of four ealves fed on a
nail Iixture of eottenseed mi. sets. out wheat than as oohti-ootoi with
anotherlotef solves fedeaacroieaistureefeora, eats. saewheat
bran. )othlsts reesivedthe shesauuntofeerusilaceamhy. rho
culves which appearod unhriftywere uller tuunths efece. lithe
sea lot too other calves ads actual pins. nose results were held to
mootothot eettenseed neal them not be fed to salves ufler to.
“of one. .

heceesotionwiththe sans investigatinabull ealfwas fedeaan
exclusive diet of settenssed asel out uni-nix. Ole-furthefapeu
efeettsnseedasalwasfedatthe startandthiss-ouetsasaaduallyin-
ereasedumil lobpoulliswas beincfed. lheealfdiedndsysafterthe
fee“. of eettenseed seal was stortod.

ni-riddie end Short (17) in mi ”ported oh investigation in uhieh
tee. fourhundredpouuddersey steers were fedoaarationofeettonseed
noel and cottonseed hulls. rho cottonseed noel was fed at the rats of
teepouflspsrdayatthestarteftheexperinentandthisanuatwasie-
ereassdte feurpoundsduriuthe sued-nth. Afterwdqsenthsea-
perilent the auinlstlost their appetite and started to lose wemt.
W of injury uted were: an unsteady an reelins gait; the defects
of leesastioa appearing in all four lilbs. llindnss secured in both

 

 

11

steers and one aninl died. he erythrocyte count of the aninl that
died was below nor-n1, s,'mo,ooo per cubic on. l'he heaoclobin who so
per cent nor-l.

more (18] in 191‘ started an investigation to deteraim whether
or not thetiuse‘of cottonseed uol and the use of cottonseed hulls were
injurious to dairypcows if fed in large amounts and if so the nature ani
extent of the injuries. three lots of five heifers each were used in
the exporinont.

not 1 received a heavy ration of cottonseed meal with tor: little
others-rain food. nay for-ed the roughage portion of the ration. lo
eottonssed hulls were fed. .

hot 11 received no cottonseed aeal but cottonseed hulls were fed
as a roughece.

hot 111 receivsd no cottonseed products.

the cows never received acre than 25 pounds of cottonseed hulls
nor are than five pounds of cottonseed neal per day.

In hot 1 which received nearly five pounds of cottonseed per day
throughout six lactations there were 14 cases of corset, one cow lost
two quarters of her udder and two others lost one quarter each; there
were three cases of retained afterbirth; one calf was born dead and
another was very weal: at birth. In hot 11 which received approxiaately‘
lopoundsof cottonseed hulls permtherewas on oaseefcaraet. em
garter of an udder was lost, and there was one abortion.

he aniulc in lot 111 empt for two slight cases of greet were
nor-n1 throughout the expoth and produced non-n chino.

4"

 

 

 

12

mo investigator concluded that tho feeding of five pounds of cot-
tonsoed noel for a considerable length of tine was injurious to the dairy
cow, causing inns-nation of the udder, difficult brooding, and probably
having a tendency to cause retention of the afterbirth. his use of cot-
tonseed “no as a roughage where eottonseed coal was not fed did not
seen to have as pronounced injurious effects. lo nention was do of
the kind or quality of hay used in this experiasnt but it was concluted
in this experimt that shore silage is fed in the winter and shore good
We unavailable in the sumor that as nch as four pounds per day
of cottonseed sisal can be safely fed to dairy cows.

it the lorth carolina Station (19) an extensive series of investi-
ptions were carried out in which it was shown that the heavy feeding of
cottonseed asal to dairy cows does not produce injurious effects if it
is fed in conjunction with hay and silogs. his series of investigations
is reported in detail under the section "moories of the cause of cotton-
seed leal Injury". i

no work of Euffhn and co-workers (20) (21) at the Iichigan station
whore'the heavy feeding of cottonseed. no.1 with . good quality of rough-
age has proven very successful is reported in detail under the sons head.

heroenlmtf-n (22) have shownthat eottenseedaeal isas lan-
tivo as linseed oil asal when fed as the principle source of protein to
lilting cows. It had foraerly been supposed that cottonseed noel was
oonstipating ia'effect.

Eon-onto 1133 m Ibelhsr's (a) report of the death of
s,cos sheep and his that who allegedto have been «not. by ooting de-
eortieated cottonseed ealoes is the earliest report noted with rent-d to

feeding sheep cottonseed products and 'the possible injury of such a
practice. '

Ibn hthausius (23) observed for several years that the uterus of
owes, which had beenfed considerable quantities of cottonseed no.1. ia-
adiately after laabing become highly infloaod, and tho sheep soon died.
It is stated that only those aniaals eating iaerioan cottonseed were af-
fected and when the use of the asal was stopped the trouble soon disap-
peared.

loser (24.) reported the death of about 100 fattening loabs after a
few days rooting on 250 greas of cottonseed no.1 used as an auxiliary
food. rho no.1 see-ed of good quality out was fed to oxen without injury.

DAM (25) reported that four nturo sheep which were started on a
feeding experiaent in which the grain ration was a aixture of equal parts
wheat bran and cottonseed .al died after nix veers of feedingo he hit-
tion was ads of the type of roughage used.

bro recent experiasnts by Jones and Dickson (26) have shown tint
one-half pound or are of cottonseed coal with cottonseed hulls as the
roughage can be fed to fattening hate for a period of 70 days vim
producing an ill effects.

W and kidney (27) reported. having fed 65 pregnant ewes throw]!
thowintor (from as to 216 days) on a ration of cottonseed no.1 .ni cot-
tcnseed halls. so iapairasnt in health or in reproduction woo observed
except for one case of blindness which occurred.

0o: (28) reports a series of investigations froa the low llexico
Station to... fattening laabs were fed on rations containing various
anunts of cottonseed aoal ani cottonseed hulls. Ehese investigations
cover three years' work which is sumrised below:

1d

50 Ianbs per hot 95 day!
let I. Dot 11. hot III. Dot IV. hot 1'.
0orn§ lb. 0.8.1.1} lb.0.8.lol§ lb.0.S.Jl. 1 lb. 0.8.11. 1 lb.
Food 0.8.“ lb. 0.8.Hulls 0.3.hlls C. S. Hulls Cans Btovor
Alfalfa Hay
Deaths lst year 0 0 l O 0
Attributed
to 2nd " 0 l 2 2 0
feeding
3rd " 0 O l l 0
total - l a 3 0
0.8.1. 0.8.11.
reed Corn Alfalfa
Stover Hay
Deaths lst year 1 0
attributed
to 2nd " 0 l
feeding .
3rd " 0 0
total 1 1

It will be seen 111st 70 per cent of the losses occurred in the lots
that were receiving one pound per day of cottonseed sisal fed with cotton-
seed hulls. mess animals became sick about 70 days after the start of
the experinsnt. they become droopy and refused to eat. the eyes often
becaas glased in appearance. After the second day of the sickness the
aninls gave indications of considerable abdoninol pain. In several in-
stances laabs which showed these symptoms were removed free the experi-
asnt and placed on alfalfa pasture or fed alfalfa hay. Recovery no coa-
plete in every case.

Post mrtea enainations of animals that died showed an accu-Ilatioa
of serua in the abdoninal cavity, inflametion and congestion in the abe-
neua‘and snll intestine. Also, congestion in lynh glands, kidneys,
and liver. rho urine was extremely acid.

l5

Erin-onto 2311M £314! Mary (10) in 1894 reported
that two pounds daily of cottonseed weal could be fed to horses with
good success. Although the feeding of cottonseed ml to horses was not
generally practiced then, no injurious results had been reported.

connell an! Kyle (15) report three instances where the continued
use of cotton seeds ani cottonseed aeal for horses and aules ultintely
had bod effects. these reports were free faraers in foxes. Other fara-
ers reported that they had used it in liaited anunts with fair success.

Durhtt (29) in a series of experinsnts at the north 0arelin Sta-
tion tonni that two pounds of cottonseed no.1 could be included in the
rations" of horses without producing any ill effects and that under lbrth
carolina conditions it was a very econoaical feed.

reqleton (30) carried on investigations in which he supplemented
the rations of working noioo with cottonseed no.1 at the rate of 1.1
pounds per 1000 pounds of live weight. Seas difficulty was experienced
in getting the aules to eat the cottonseed asal, but its inclusion in
the ration had no effect on the health, spirit, or endurance of the ani-
nls to which it no fed.

3.11 and viniono (:1) conducted on sxperiaont in which they fed
cottonseed asal to nine work horses in a grain aixture .of cats, bran,
and cottonseed aeal. the roughage used was oat hay. Seven teal ntes
of these horses received the sons ration except that no cottonseed nal
was'fed. rho ononnt fed varied oonowhot. on. pout daily for every
1000 pounds 11.. weight was founl to be tho noot satisfactory level.

D reproductive troubles were noted in the nres which received the cot-
tenseed asal. rhey becsas safe with foal readily and dropped nornl
healthy foals. ‘

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16

One nre that received three pounds of cottonseed aeal died after
a days on the experiaent. Post aorten enaination showed an inflaaed
condition of the stench and intestines. These indications seeaed to
point to cottonseed aoal as the causative agent of the injury.

It was noted that the acres thrived better and ate their ration con--
taining cottonseed no.1 nor. readily on pasture than they did when they
were stall fed.

flaunts _w_i_t_h 215; 0urtis and 0arson (52) in 1892 reported
that during trials tho year previous tho fatalities due to feeding cot-
ton seed were nu-rous. Six to seven weeks after the pigs were placed
on trial they died of cottonseed aeal injury. Boiling the raw seed be—
fore feeding seencd to reduce the toxicity soaewhat as indicated by fewer
deaths. they describe the syaptoas as follows: The firet sign of sick-
ness appearing in free six to eight weeks after cottonseed asal is added
to the ration, is a noping dullness of the aninl with loss of appetite
.and tendency to lie apart. Iithin the course of 12 to 56 hours, often
within shorter tins, the aninal becomes restless, stoggering in his gait;
breathing, micron and spasmdio; bare skin showing reddish inrionnotioh;
sight defective, and both nervous and moular systene feeble and abnornl
in action. he fatal cases all show ”thumps”, spas-odic breathiig, am
in now instances the aninl will turn in one direction only. rhooo pigs
which do not die beoone stunted and do not grow.

Emery (10) reports a case where two pigs which had received skin
nilk were placed on a cottonseed steel experiasnt. One pig received cot-
tonseed noel and wheat bran, the other received corn noel and wheat bran.
After six weeks on these rations the cottonseed meal was increased to two

pounds per day and the pig receiving this amunt soon lost appetite and

I."

refused to eat. Replacing the cottonseed meal with corn resulted in
the pig's recovery.

Oarrcevin (as) observed that hogs which were fed Egyptian cotton-
seed and oottonoooo cake were poi-choc very severely. no injected a
cold water extract of the seed into a dog and caused death in 18 hours.
He observed that the kernels were lich are poisonous than the hull an
that coneroial cottonseed oil was her-less.

connell and Kyle (13) in 1899 reported the experience of a number
of Iron. farmers oho had fed cotton seed or cottonseed no.1 to hogs.
Ihero uncooked cotton seed was fed in dry lot the results were in alnst
every case disastrous. The only favorable results which were reported
were in cases where cooked seed was used or where the hogs were allowed
to run on pasture.

Diaiddie (:4) fed cottonseed coal in combination with corn, with
bran, with ma turnips, and with bran, corn and cow pea hay. rho-o.
pigs receiving cottonseed noal one part and corn three parts, died after
so days. Inch pig at the ties of death had consuasd 25 pounds of cotton-
seed nal. The ration having turnips in addition to corn and cottonseed
nal did notprove successful. where bran was substituted for corn there
no a nrked inrcvenent. 'hore bran was fed with cottonseed aoal as
nob as 1.d pounds per day was fed without producing haraful results.

A sew was fed for the last 80 days of her gestation period on a ration
containing cottonseed aeal. She received 1.3 pounds per day withut
bra to herself or her litter.

crude cottonseed oil no fed to three pigs for so weeks in anunts
exceeding that which is contained in a fatal ration of cottonseed asal.
D haraful effects were noted.

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18

Characteristic post aorta s, findings ; of pigs dying fro. cotton-
seed-eel injurywere inall oasesanmte dropsyofthepleuralam
heart sacs with intense congestion of the liver and kidneys. no 1.“-
ate cause of death was thought to be due to suffocation from coqressien
of the lungs. ‘

coorgoon, hurtio em Otis (so) used cottonseed neal with corn seal
as one-fourth and one-half'of the ration. The results in both cases were
disastrous, the pigs dying in fron three to eight weeks after going on
trial. are larger pigs seemed the sure resistant. In all cases poet
norten enaination revealed severe influnation and cemestion of the
intestines, lungs, and heart. Up to the tine that the eysptcns of cotton-
seednsal injury set inthe gainshedbeenverygocd.

curtis (:6) fed cettonseed seal with corn and ceb neal and butter
ailk to pigehweighing about 100 pounds. the pigs eeno good gains for
about sinweeke and thenbeeans fatally sick. Ineverycase the cunt
of cottonseed aeal consul-ed before deathceccurred was fron 8? to 88
pounds. rigs following cattle that were fed fron four to seven poems
of cottonseed noel showed no injurious effects in .17' weeks, the duration
of. the trialo ‘

hear (a?) founi that .25 to .61 pounds of cottonseed neal daily
per 100 pounds or live weight caused sickness one death in pigs in five
to seven weeks. Yellow corn formed 75 per cent of the ration. . Green
pasture tallied to alleviate the toxicity sonewhat.

hrshell [88) reported eneriunts in which he lined cottonseed
wool with cornchops aul after adding water allowed the nixture te fer-
.nt until it was thoroughly sour. he feraented nixture was fed to
fattening pigs weighing fro. 100 to 130 pounds. Where the ration con-

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19

tained one-third cottonseed no.1 the pigs did well fer 74. days. After
that tine one anisal died and one other refused te eat well. One lot
which received fermented cottonseed steal as one-half of the ration did
well forGOdaysbut after that ties threehogscnt of tendiedandan-
other bacon chronically sick and lost weight. Up to seven pounds of
the nixture of corn and cottonseed neal was fed daily, or 3.5 pounds of
eettonseed. It was concluded that fer-eating cottonseed reduces but does
not entirely elisinate its toxicity.

Curtis (59) red fermented cottonseed Deal to pigs as 80 per cent of
the ration for 9. days with very good results. After that ti... the gains
were not as good as they had been previously but the auifils apparently
renined in good health for a aonth longer, when the trial was finished.
Ln average of .92 pounis of cottonseed seal was fed during the entire
period.

talker (40) in 1916 reported that the addition of copperas to a ra-
tion of three parts of corn to one part of cottonseed seal prevented in-
M to piss.

Iithers am Brewster (a) had previously shown that iron was effec-
tive as an antidote to cottonseed sisal poisoning in rabbits.

Withers and earmth (42) later reported that the addition of iron
in the fore of r. e12 or copperas to a ration containing cottonseed seal
greatly reduced the fatal effects. Out of a total of is pigs getting
the iron, four died while out of 19 not getting iron 13 died.

Renal and Yedder (43) in 1915 and tells and lwing (4A) in 1915
reported evidence to show that cottonseed no.1 injury in pigs is due to
the feeding of a deficient diet. ‘l'hese investigations will be reviewed
later in the section ”rhecries of the cause of Cottonseed lbal Injury.”

20

Goldberg and Maynard (45) reported an experinnt in which 12-pigs
previously innoculsted against cholera weighing froa 50 to 50 pounds
each were fed on a ration nude up of 60 per cent corn noel, 10 per cent
wheat aiddlings, 25 per cent cottonseed neal and five per cent mlasses.
d aineral ainture was fed "ad libitun".

sin. of the pigs died "within eight to 12 weeks froa the beginning
of the trial. the usual synptoas observed were failure to eat; vomiting;
rapid, short shallow breathing; aneaia, and a weakness of the leg uncles.
i very coqlete post sorten report was included in the investigations.
Particular enphasis was placed on a study of the lesions found. rhey
found a generalised edeaa throughout the body. he initial lesion in
cottonseed steal injury they concluded is ascites.

Barnett and Goodell (as) fed three pigs a ration of two parts corn
to one part cottonseed no.1 for as days without producing any noticeable
injury. 8.8 pounds of cottonseed nal was consund daily per pig.

Warren and Iilliels (‘7) fed cottonseed no.1 with lilo chops an

with silo chops and tankage for a period of 120 days. In one lot of
nine pigs where cottonseed neal foraed 11 per cent of the ration there
he one death froa cottonseed meal injury on the 70th day of the experi-
nent. In other pigs showed no smtons of inJury at any tine.

nhstedt and ee-werkers (49) at the Ohio Station reported that a
pig roooiriog 20.2 per cent oottonoood nal in its ration died on the
180th day of the experiaent. he pigs receiving 25 per cent cottonseed
seal died on tn 67th and 78th days of the enperiaent. Post mrtea en-
asination in all cases. showed nrked henrrhegic condition of the ass-

eatery.

2].

halo (as) has reported a series of investigations on cottonseed
aeal for swine which have been carried on at the lens Station. It is
concluded fron the results obtained that where not acre than nine per
cent of the ration is node up of cottonseed noel it can be fed safely
te brood sows, boars, growing pigs, and suckling pigs without producix
any harnful results. cottonseed aeal fed at a 15 per cent level resulted
in a loss of three pigs out of 20 which on post aorten enaimtion showed
tn characteristic sylptons of cottonseed sisal injury. There were no
losses fron cottonseed nal injury in a lot of 20 pigs fed a ration con-
taining 12 per cent cottonseed seal but they did not look as thrifty nor
nke as good gains as pigs which received only nine per cent cottonseed
seal in their ration. Ground raw cottonseed proved very unsuccessful
when used as a protein supplement for 34 pound pigs. in 4o per cent and
as 25 per cent of the ration it proved unpalatable and caused scouring
on the 18th day of the experinent. After so days on the trial the pigs
were round but two of the five pigs died froa cottonseed poisoning on
tn 86th and 28th days after the start of the experinent.

Minute with M followg steers receivg cottonseed seal ’
deergson, Burtis and Otis (35) report a case where do pigs following steers

 

fed en cottonseed noel died in six or seven weeks showing the seas eyep-
tons of poisoning as those which were fed cottonseed nal directly.

Settle and Iain (50) reported that of 12 hogs following steers whieh
received 3.23 pounds ”of cottonseed neal per dq, one died. he cause of
death was not definitely found to be cottonseed aoal poisoning, however.
no reaaining eleven node fairly good gains over a period of 180 days.

22

arisen“ with Poultry Roberts and Rice (51) in 1597 observed
that the eggs from hens fed on a nitrogenous ration one-third of which
was cottonseed noel were .11 in sise, had a dork colored yolk, and a
red watery albunin. The eggs were also observed to have poor heaping
qualities as compared with eggs fron hens which were receiving a car-
bonaceous ration which contained no cottonseed aeal.

Moon (52) fed cottonseed meal to laying hens as 50 per cent of
a ration eonposed of cats, bran, and cottonseed neal. rho health of
the hens was not affected as far as could be seen. About eight per cent
of the eggs from hens in their second laying year were found to have cot-
tonseed seal spots. Eggs froa pullets were not affected.

Ehe cottonseed neal spot is described so, ”a brown spot with a red-
dish tinge varying fron the sins of a pin point to a quarter of an inch
in dianter. he spot floats on the surface of the yolk and can readily
be seen when the egg is candled. Often there are several spots in one
egg. rhe fertility and hatchability of the eggs are mt affected.”

It was concluded fron this investigation that laying hens are not
injured by the consumption of fairly large onount. of cottonseed no.1
althouh it is not reconnended that it be fed as the sole protein sup-
pleaent.

Sherwood (63) fed laying hens cottonseed Ilal in varying oaounts
and studied the storage quality of the eggs produced. lone cf the eggs
showed any serious discoloration of yolk or white when first laid all
little. or no change in color was noted until after four weeks in cold
storage. After four weeks in cold storage the eggs free hens receiving
fl and 31 per cent cottonseed aeal in their nah began to deteriorate

23

in color. lggs froa hens receiving nine por cent cottonseed no.1 in
their nah secaod to keep a good color in cold storage. lilo and kafir
were fed in addition to the such in these lots. Where on all nah ration
ns used the presence of nin or 12 per cent cottonseed seal cnsed a
deterioration in the color of the eggs kept in cold storage after four
weeks. When cottonseed and constituted three to six per cent of the

all .nehretion the color of the eggs roneinod good for as long as so
weeks in storage.

in feeding of a fresh, succulent green feed will inrove the star-
age qualities of eggs where hens are fed a ration containing a fairh
large aunt of cottonseed nal.

Erinnts will}. 2.1: Sync and Gardner (54) in 1915 in connec-
tion with their study of the toxicity of sodiun pyrophosphate found that
cottonseed aeal proved fatal to rats when fed as an exclusive dict; even
five grass of cottonseed noel per rat per on when supplcaented with cats
and bran were fatal. no.1 which no partially extracted with alcohol and
ether ns found to be such less toxic although death did result after a
few weeks.

nocoiiun, Sin-end. end Pits (55) while studying the vita-in i con-
tent of several vegetable oils round'thet other extracted cottonseed oil
no toxic to rats but conercial cottonseed cil extracted by hot pressing
was not injurious. Cottonseed oil was found to be very low in its vita-
lil A content.

Richardson and Green (56) fed rats rations containing froa 33 to 70
per cent of cottonseed seal and cottonseed flour. to cottonseed seal
poisoning was produced altlnudt son of the anisnls died, probably fre-

24

indcqnatc diets. The addition of butterfat to a diet composed of lard,
starch and cottonseed did not improve growth. It is probable that the
diet used was inadequate in ninerels because no nincral supplement was
used and the addition of whole silk powder produced norunl results. '

Richardson and croon (57) in a later paper reported that diets eon-
posed of 50 per cent cottonseed flour, supplenented with protein free
silk and butterfat produced nornl growth. lo toxic effect ns observed
when this ration was used for four successive generations; reproduction
and growth were normal.

Osborne and Mendel (58) fed cottonseed seal and cottonseed flour in
a ration which contained inoddition starch end lard. no indurious re-
sults were noticed and in one case a norml litter was reared. In several
eflthe successful cases butterfat was added as a supplcnnt and improved
pins were noted. hw cottonseed kernels were used in the rations for
rate with the result that the rats died in from six to 12 days. In a
series of experiments'whore the kernels were subjected to different treat-
nnts it was found tnt steaming the kernels for two hours destroyed the
toxicity while heating in an electric oven at 110° C. or steaning for one -
hour did not.

Ravens (59) used 23.9 per cent cottonseed seal in an adequate diet
for rate over a‘period of seven weeks. flu symptoms of toxicity were noted.
Gallup (60) fed cottonseed seal at various levels in rations for
rats, in determining the digestibility of the cottonseed protein. 09 to

51.5 per cent of the ration ns cottonseed noel. No injurious effects
were noted. the addition of gossypol to the rations did not lower the
digestibility.

Bethke, hohetcdt and co-workers (48) fed cottonseed seal to. rats

as 24.2 por cent of the ration and observed no injurious results.
theories of the Cause of Cottonseed Heal Injury

Since Voelkcr (4) in 1873 published his report on the injurious
effect accruing iron tho feeding of cottonseed seal, investigators have
studied the effects of cottonseed seal feeding and a miner of theori.
and speculations have arisen to account for the injury which has oeso-
tises occurred in connection with its use.

Choline g Betainc 351m (51) node one of the earliest .ttenpt. to
solve the probles of ”cottonseed seal injury”. He isolated a large pm-
aration of choline fros an alcoholic extract of a cottonseed cake which
had been fed to young cattle in Cox-saw with fatally injurious results.
He concluded that there no but one alkaloidal substance present in cot-
tonseed cake.

kitthansen and wager (62) one year later in 1882 isolated betaine
fros cottonseed cake. I ‘

Oochtgens (63) bed previously shown that .3 gran of choline injected
into . vigorous oat had caused an inedioto paralysis and that .s gran
ns sufficient to cause instant death. he believed that betaine ns
non-poisonous.

knell (64) showed that betaine is present in such larger shunts
than choline in cottonseed coke and that together they constitute about
.24 per cent of the cake. .

m Hoeslin (65) in 1906 reported a series of experissnts which

showed that when choline is fed by way of tho .nuth its toxicity is very

liaited. Even chddlin hydrochlorate which was found to be sore toxic
than chloline, could be given in two gran doses to a rabbit without
producing serious results. .

rho possibility that choline my be converted to sore toxic sub--
stances such as neurin or suscarin has been suggested. Prienn (66)
reported that ptomaine substances are probably round frcs tho nitro-
gen containing components of lecithin in cottonseed seal. he also sug-
gested that the unsaturated fatty acids present in the fat extraccd frca
cottonseed exert sose influence on its toxicity.
Parasitic Orflsm and; Decomsition Products of. Bacterial £1133 Von
lathausius (23) attributed the toxic effect of cottonseed seal to parse-
sitic organisms associated with the seal.

ndgorton and llorris (57), however, round that seal infected with
the solds that usually grow on cottonseed were even less toxic when fed
to rabbits and guinea pigs than anal sade fros uninfected seeds.

nnig, Spieckernnn an}. Olig (6e) nde a study of the deco-position
products formed by various adore-organisms working on cottonseed seal.
rhey found that all of the organisss investigated gave oisilar physio-
logical effects and were affected by the conposition cf the meal and the
air supply. 1 number of cleavage products were caused by the bacteria
which attack the local, cone of then being toxic but at n ti. during
the decosposition were the injurious substances present in Mficicnt
amunts to nnifest themselves in physiological symptoms.

High Protein Content. rictse (9) investigated several cases of young
calves which were thought to be poisoned by cottonseed meal feeding.

Phase calves received three heaping liters of cottonseed seal per day

27

in addition to skim milk, hay, and linseed cake. He believed the injury
was due to the high protein content of the cottonseed cake.

It has also been supposed by so- that cottonseed Dal injury was

caused by its fiber content and also by the oil it contains.
Phosphoric £333. Hardin (c9) in 1892 advanced the theory that the pres-
ence of nut. and pm phosphoric acid which he found in the aqueous solu-
tion of the meals he examined accounted in part at least for the injurious
effect which occurred froa feeding cottonseed steal.

Orelford (70) continuing the work of Hardin, by a number of pharma-
cological tests on rabbits cans to the final conclusion that the chief
poisonous property of cottonseed meal in a salt of pyro phosphoric acid.
The fact that some meals were more toxic than others was thought to be
due to the conversion of ortho into the meta and pyro phosphoric acids
by heat during the process of manufacture.

Edgerton and llorris (6'!) two years later presented evidence to show
that pyrophcsphoric acid had nothing to do with cottonseed neal injury.

Anderson (71) isolated the organic phosphoric acid in cotton seed
which he found-to-be similar to phytin but he found no evidence to show
that it is the poisonous principle present in cotton seed.

'ithers and Bay (72) found that a pepsin pancreatin extract of cot-
tonseed was not toxiohto-rabbits in the amunts in which it is usually
fed and tlat the residue was hrs toxic than the extract. They also
showed by feeding pure sodiua pyro phosphate in aaounts corresponding
to that in the seal that it was not the substance responsible for the
injury and they further proved that the toxic effect my occur in the

ual when the pyrophosphoric acid salts are not present at a toxic level.

28

Sync and Gardner (54) later corroborated the work of Withers all!
Bay when they administered sodiua perphosphate in food to rabbits, rats,
and cats. The tolerance of rats for sodiu- pyrophosphate was seven time
the aunt found in the cottonseed steel which had proved fatal.
Acidosis Forbes (73) found that in 100 grans of cottonseed neal the
excess of acid over‘base for-ing elements was equivalent to 7.7 cc. of
ncrnl acid.

wells and Ewing V4) believed that cottonseed neal injury night be
due to acidosis for they found an excess of acid over base forning ele-
nents in 100 grams of cottonseed seal equal to 8.2 cc. of mml. Later
work by these investigators on swine did not substantiate the theery and
they cans to the conclusion that acidosis is not the cause of cottonseed
seal injury.
3.9.9.9. Principle 2111?. Protein Elecule withers and Brewster (41) in
1913 put forth the theory that the toxic principle of cottonseed neal
was a constituent group of the protein soleeule containing loosely bound
sulphur and that the toxic effect of the .al was due to he action of
this group upon the iron of the blood. The addition of iron in the fern
of iron citrate to the rations or rabbits receiving cottonseed weei al-
leviated the eynptons of injury and when fed before the smtons appeared,
prevented their developnent, although the rabbits ate five time the
eaouet ef nal which proved fatal to rabbits which received the unsupple- ,
mated ration.

withers and Garruth (42) in a later investigation used pigs as en-
perinental subjects. Iron in the fora of re cl: and copperas was used.
Out of a total of lo pigs getting the iron in addition to a ration sen-

taiaing cottonseed .al four animls died while in a lot of is which did

29

mt receive iron 15 died. It is suggested that iron salts facilitate
oxidation of the toxic substance. '
Deficiefl Disease Theories wells and swing (4.4) using .1. week old
pigs, fed cottonseed meal in a ration with starch, sugar and ski-ed Iilk.
Ihen cottonseed meal was fed at the rate of 25 grains per kilo of body
weight per day with five cc. of skim ailk, death resulted in free 30 to
50 days. When gluten flour replaced the cottonseed steal the result was
also fatal. The addition of ‘70 cc. of skin silk a day assured Iaintenance
am a fnir enount of growth. hilure of both cottonseed mal and gluten
flour on a ration balanced fron the standpoint of carbohydrates, fats,
and proteins was held to indicate a deficient diet rather than the pres-
ence of a_ definitely toxic substance.

vitamin e noun-1 and redder (45) in 1915 advanced the theory that
the cottonseed seal injury noted in pigs is a deficiency disease analogous
to the disease known as beri beri in nan, if not identical with it. They I
beseltheir hypothesis upon the sinilarity in post norten findings and up-
on experiaental work with pigs. They fed pigs upon polished rice and a
disease resembling beri beri in can developed. The post norten era-imp
tions on these pigs showed a great similarity to those of pigs which had
died of cottonseed seal poisoning. They point out that acute "injury"
corresponds to wet beri beri in non and chronic injury corresponds to
the dry forn in nan.

Withers and Garmth (75) were unable to substantiate the work of
Ronael and Yedder. Pigs to which they fed a ration of cottonseed neal
supplemented with milk and green feed died, while pigs on cottonseed

kernels free which gossypol had been extracted with ether developed

normally.

30

Bechdel, Eckles and Palmer (76) reported that the requirement of
the dairy calf for vitamin B is either very small or that the vitaain
is synthesised by the animal.

lechdel and co-workers (77) have since shown that vitamin B can be
synthesised in the neon of the cow, and a cow on a vita-in 3 free ra-
tion does not develop smtoae which result from a vitamin D deficiency.

Easy and llsndel (78) reported that cottonseed neal contained seas
vita-in 3 but Itch less than is found in yeast.

’ Richardson and Green (79) found that cottonseed heel furnished a
considerable aunt of the water soluble vitamins.

Stevens (80) in recent work has shown that cottonseed seal is an
excellent source of both vitaain B and vitamin G. Ten per cent cotton
seed seal in a vitamin 3 free ration contained enough of both factors
of the conplex to produce approxintely optin- m

iron Deficiengy licGowan and Christen (81) have advanced the the-
ory that cottonseed meal injury is associated with an iron deficiency
in the ration. They found that the entons ef poisoning are siailar
te those which occur in suckling pigs on iron deficient diets and they
believe that the injurious results usually occur in pigs which have saf-
fered in the suckling stage froa an iron deficiency. They added iron
in the fore of ferric oxide te pigs on a ration containing a lame shunt
ef cottonseed ual and were able to delay the onset of the injury.

halverson and Sherwood (19) have reported work free the North Oars-
line Station which shows that the addition of iron to the ration in the
fern of copperas used at a .5 per cent level failed to show any benefit

in delaying the onset of the symptons of cottonseed neal injury.

31

Gallm (82) reported that iron salts delayed the onset of cottonseed

weeiinjnryinewine. '_ _ ~

_. m 93; rectors carried Bl. gel need, Hurt-en and Addington (so)
in 1928 showed that a close relationship exists between the symtoss of ‘
cottonseed seal injury in cattle and the injury produced when too Itch
concentrates are fed in proportion to roughage er where a roughage of
poor quality is fed. They produced a condition of blindness and stiff-
ness ef gait in two calves on a ration of wheat straw, as the roughage,
and corn gluten and corn distillers grain as the chief sources of pro-
tein. lo cottonseed meal was fed. calves receiving cottonseed seal as
a chief source of protein with wheat straw as a roughage developed the
ease symtons of injury, later having convulsions. The anisale died at
875 and 350 days of ago. Where tinthy hay and silage sore fed as a
source of roughage heifers receiving cottonseed seal as the chief source
ef protein developed nornlly.

mm and Moore (21) continuing the work just cited, fed fiwe oews
which had been raised treeeo days of age on a ration having cottonseed
ual as the chief source of protein, on a ration of tisethy hay, corn
silage, yellow corn, and cottonseed seal during tee lactation periods.
cottonseed nal was fed in sufficient enunts to meet the entire protein
requiresents for asintenanoe end lactation. is Itch as 11 pounds per day
was fed during the early mnths of lactation in the case of ease of the
cows. Ming the first lactation two of the cows received an average ef
over seven pounds of cottonseed sealvper day throughout the entire period.
These cows produced approxisstely 10,000 pounds of silk in 10 nnthe.

32

[one of the five animals developed any symptoms of cottonseed seal in-
jury. There was no sore udder infection in the case of the aninals fed
cottonseed meal than there was in the case of animls fed on linseed ail
ual. A bull calf 90 days old fed cottonseed from the same lot that was
fed to the cows, but getting wheat straw as a roughage deve10ped secure
when about 270 days old.

is a result of these investigations Huffman and his associates can
to the conclusion that cottonseed meal injury in cattle is due to a lack
of a factor or factors carried by hey.

it the North Carolina Experiment Station (19) a series of investi-
gations on cattle were started in 1915, which have recently been sus-
nrioed by Halverson and Sherwood (19).

In the) early trials it was found that when cottonseed seal was fed
with corn silage loss injury was observed and better gains were made
than when cottonseed hulls were used as the roughage. Also a limited
assunt of pasture fed with cottonseed meal and hulls produced a fair
shunt of growth. The addition of capperas to the ration at a 0.5 per
cent level and the substitution of 50 per cent of the cottonseed seal
for corn did not relieve the symptoms of cottonseed meal injury as in-
dicated by unthriftinees and impaired vision.

In 1920 a more extensive investigation was started which included
trials on 20 cows. Rations restricted to cottonseed meal and cotton-
seed hulls resulted in the development of symptoms of cottonseed seal
injury in approxintely the same time in all cases. Characteristic
mtoss which developed were: impaired appetite, staggering gait, af-

fected eyesight (in many cases blindness), and spasms.

 

..

 

The substitution of 50 per cent of the cottonseed meal by 50 per
cent oats did not alleviate the injury.

When the roughage contained corn silage or silage am corn stever
the synptons of injury were much milder and spasms an! total blindness
did not occur. When beet pulp was used the symptoms were similar to
those which developed when cottonseed hulls furnished the roughage por-
tion of the ration.

Various supplements were added to the basal ration of cottonseed
Ieal and cottonseed hulls after the sylptoms of. injury had set in.

fhe addition of yeast at the rate of one pound weekly to a cow suf-
fering from spasms and anorexia resulted in a marked inprovensnt. Ber
appetite improved and no mre spasms occurred. She gained steadily in
weight for 278 days. Another cow in a critical condition was fed two
pounds of yeast daily and recovered sufficiently to produce a living
calf. it the. tine the yeast was added to the ration the cow was re-

ceiving 0a Go and butterfat in addition to the basal ration but prob-

3
ably they were present in insufficient amounts.

the addition of casein as 9.93 per cent of the ration did not al-
leviate the injury.

the ration was known to be very low in its calciun content but the
addition of ca do: alone in amunts as high as 2.5 per cent of the ration
was not beneficial.

Alfalfa leaf seal of excellent quality fed as five per cent and as
10 per cent of the ration did not adequately supplement the ration.

Butternt fed as 1.94 per cent of tin ration did not prevent the in-

jury even when the calciun content of the ration was nde nornl.

34

By using the preventitive treatment (adding the supplemnt before

injury set in) the amounts of supplements and the combinations needed

to prevent injury were obtained.

be Approxintely Adequate

They are sumarised below.

Basal Ration - Cottonseed Real and Cottonseed nulls

Per cent in Concentrations

Supplements Used in cottonseed Bel Rations Which Proved to

 

Ration Calciun Steamed Butter Cod Liver Yeast Iheat Crude
lo . carbonate lone Real fat 011 Hubris Casein
17 £24 - - 1.99 6.42 - -
16 2.92 - - 1.94. - 9.72 -
13 1.96 - - 1.94 .87 7.81 -
10 1.92 - 5.4.1 - d.26 6.26 -
10 a 1.80 - 8.21 - 6.01 8.02 -
9 1.92 - 1.71 1.71 1.71 6.82 8.52
Basal Ration - Cottonseed Neal and Silage
11 2.10 - 1.86 - 1.86 - 9.32
12 - 3.48 - 1.74 4.55 6.85 -
Basal Ration - Cottonseed Bal, Corn, C.S.Hulle, Silage and
_ Stover
22 2.89
23 2.75

 

It appears that all of the rations which were adequately supplanted

contained approximately two per cent or more of calciun carbonate, six

per cent or are of yeast or wheat embryo and nearly one and three-quart-

CV

'
(*1
I,

 

35

ere per cent of cod liver oil or three and one-quarter per cent of
butterfat.

In a are recent investigation in the series it has been shown tint
feeding of meals from other high oil bearing seeds other than cottonseed,
with poor quality roughage resulted in the development of the same sm-
tons in dairy heifers that characterize cottonseed neal‘injury. Linseed
oil meal, peanut oil meal and soybean oil meal were used with wheat straw
as a ration for dairy heifers. a staggering gait, swollen legs and joint;
blindness and convulsions developed in about the sane length of time that
they did when cottonseed meal was fed with wheat straw.

Vitamin A assays of the livers of heifers which died on these rations
showed that.5605 grm.of dried liver per day did not contain a sufficient
aunt of vitamin A to cure opthalmia in rate. a similar amount of dried
liver from normal animals was effective in curing opthalmia.

a similar assay showed that best pulp and cottonseed m1. fed .t
the rate of one gr. daily to rats affected with epthalaia contained in-
sufficient vitamin A to improve the condition. Wheat straw sue found to
have a limited amunt of vitamin A.

In a recent project with milking cows it has been shown that when
the ration consisted of one-third cottonseed meal and a mineral eixtsre
and two-thirds roughage made up of eqml parts of cottonseed hulls, corn
silage and alfalfa hey the cows suffered no ill effects over a period of
176 days. However, mien dried beet pulp repldced the silsge, poor health
an}. failure in five of 10 lilting cows resulted.

In discussing these results Halverson and Sherwood concluded tint
cottonseed meal injury is due to a deficiency in the ration and not to

the presence of a toxic substance. i'hey believe that the data suggests

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86

a deficiency of vitamin d and inadequate calcium. hey do not account
for the improved results which were obtained when yeast was fed. i'hey
also suggest that vitamin D my not be present in sufficient amunts to
insure good calcification of bone am teeth.

Bechdel, Honeywell, and butcher (83) described the symptoms of vi-
tamin a deficiency in heifer calves which had been on a ration low in
vitamin A for six to seven months. Eden appeared first in the legs of
the animals and then gradually spread to the neck and shoulders. One
heifer went comletely blind. ani another had fit like spasu. he calves
were born prematurely to the heifers on the deficient ration. Post
mrtem examinations showed the tissues were infiltrated with a watery
fluid in the region where edema existed. these entoms agree fairly
closely with those'which occur in cottonseed meal injury.

Jones, Ecklee, and Palmer (84) described the symptoms of vitamin
A deficiency in calves as loss or appetite and failure to grow, later
scouring developed. Zerophthalmia set in and respiratory troubles oc-
curred. Post mortem findings revealed edema of the kidneys, atrophy of
the intestines, necrosis of the rumen and sclerosis of the liver. These
investigators found that wheat straw is a fairly good source of vitamin
d as indicated by curative treatments with rats.

lloCollum, Simmnds and Pits (55) have reported that cottonseed oil
is very low in its vitamin 1 content. Alfalfa leaves were found to be
an excellent source of this factor, however.

Steenbock and Cross (86) found that five per cent of inmnature a1-
falfa leaf added to a vitamin L free diet had sufficient vitamin a to

promote nornl growth and the rearing of some young.

37

In view of the high vitamin d content attributed to alfalfa and
also because of its high calcium content it is difficult to explain the
failure of the five cows on the lorth Carolina experiment whose ration
censisted of approximately 25 per cent alfalfa hey.

Cosml Iithers and Ray (85) in 1912 extracted cottonseed Dal
with gasoline and then further extracted it with a hot alcoholic solution
of la 03 for two hours. The extracted meal when fed to rabbits at the
level which caused death in the see. of the unextracted cottonseed meal
did not produce any apparent injury although the rabbits lost weight.
hey concluded that the beneficial effect was due to hydrolysis, to the
formation of a sodium salt, or to some other change not yet determined.

Iithere and Carruth (87) two years later separated from cottonseed
a substance which appeared identical with one isolated from cottonseed
by firchlswski in 1899 which he had nanmd goseypol. It was administered
to rabbits in various ways and was found to be toxic in every case. my
found as did lurchlewski that gossypol was readily oxidised in an aloe-
belie solution of neon, the oxidation apparently rendering it non toxic.
his fast would explain their earlier work.

Iithers (88) in 1915 published the results of a more extensive in-
veetigaticn on the toxicity of gossypol. new cottonseed herneis were
extracted with petroleum ether (in which gossypol is insoluble) an! the
extracted kernels were fed to rabbits. Fatal injury resulted. Kernels
of raw cottonseed which had been extracted with petroleum other were
further extracted with ethyl ether, ani the residue which anunted to
about 2.5 per cent of the original weight of the nmterial was administ-
ered to rabbits. It was found to be toxic when injected intro. peri-

 

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38

toneally when fed in small doses or when fed through a catheter. Death
occurred in the case of injection in three to 15 hours. In the case of
catheter feeding in 12 - 25 hours and when fed orally death occurred in
from eight to 15 days.

Crude gossypol acetate precipitated from an ethyl ether extract by
addition of glacial acetic acid was found to be more toxic than the crude
residue from other extraction.

Oxidising the gossypol acetate with alcoholic UH apparently cem-
pletely eliminated its toxicity.

Characteristic post mortem examination of the rabbits which died
from the injury showeds reddish serum in the abdomiml cavity, liver
congested, lungs slightly congested and endanmtous. more was some in-
testinalinflanmation and some hemrrhage at the pyloric and. The kid.-
neys were also somewhat congested.

Schartse and dlsberg (89) ends a study of the toxicity of gossypol
en cats and rabbits. Metabolism studies with cats showed that they were
often in negative nitrogen balance when on a ration of meat, butterfat
and whole milk powder. ninety mgm. of gossypol were administered daily.
Gossypol was noticed to have a paralytic effect on cats and rabbits not
merely upon the neuro miscular apparatus of striated mscle but also up-
on that of smooth muscle. It was also found that gossypol is a circu-
latory depressant. It produces a fall in blood pressure iueedieteiy
after injection. tor scum tine after injection the heart shows shipped
or weak beats.

Post mortsm examination showed that edema my fellow gossypol injec-

tion as well as gossypol feeding. There is local edema of the lungs

 

 

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39

which my be hemorrhagic and there is also effusion in the serous
cavities.

Schwarts and Llsberg (90) working with rats found that the thresh-
held of toxicity for gossypol is about .0675 per cent of the ration.
Reproduction at this level was poor, however. above this level tr. in-
cidence of death was large. They found that in general the toxicity of
cottonseed is indicated by its gossypol content but in a few varieties
the toxicity in biological assays was found to be less than tin gossy-
pol content would indicate.

lemul (91) administered gossypol through the diet am feursl that
it is very slowly absorbed. In rabbits death was produced in about 14
days when .1 gram per day was fed. When introduced directly into the
blood stream the effects are noticeable at once and death results quickly.
Stall quantities of gossypol mixed with fresh blood. reduced its oxygen
carrying capacity aaterially. Gossypol was also found to exert a hen-
lytic effect on the erythrocytes.

'l‘he anatoaical effects of tin poisoning were found to be a passive
hyperenia and edema of the lungs and son hydre thorax. Intestinal in-
flamtion was noted in rabbits fed the free gossypol.

Vithere and Oarruth (92) compared the toxic effect of gossypol and
cottonseed products in rats, rabbits, chickens and swine. Rats were fed
a ration containim up to 50 per cent cottonseed usal without producing
injurious results. Long coched cottonseed steal and ether extracted rw
kernels were found to be practically non toxic. Vell coolned cottonseed

nal with milk as a supplement proved very successful.

 

‘Iull‘i

40

Rabbits were found to be much more sensitive to injury than rats
and were poisoned by long cooked cottonseed meal which had been foum
to be non-toxic to rats.

Hens fed on cottonseed meal had a diminished egg production when
fed excessive amounts of seal but they sustained no other noticeable
him. When ru kernels of cottonseed were fed a discoloration of the
egg yolk was noted.

’Pigs were found to be very susceptible to cottonseed meal injury
and were poisoned even by thoroughly cooked cottonseed neal fed at 25
per cent level with cracked corn nus wheat bran. Soybean and and pea-
nut sisal fed at the same level produced good gains. Supplenenting long
eooked cottonseed meal and the basal ration with butter, asatscrap, milk
powder, and a salt mixture did not prevent death. These investigators
a. to two conclusions: that there is marked species difference in
susceptibility to gossypol poisoning or "gossypol tolerance" as they
call it and further, that in the cooking process the gossypol is con-
verted to a less toxic substance which they call D-gossypol,

'ithers and Garruth (93) have suggested that while most of the goe-
sypel disappears in the cooking process due to an oxidation sees nay be-
eone bound through condensing with amino and carbcxyl groups of protein
-lecules.

clerk (9d) furtlnr elucidated this hypothesis. During the heating
and pressing to which the seeds are subjected the resin glands contain-
ing the gossypol are disrupted and possibly much of the @ssypol is dis-
selved in the oil present. It thus cones in intintc contact with the

protein of the seeds and in this condition favored by heat and pressure,

probably condenses with free amino groups of the protein mleculcs as
it does with mny primry amines, forming substances similar in type
to dianiline gossypol (which is readily obtained by condensing pure
gossypol with aniline).

clark (95) has shown that aniline D—gossypol is identical with di-
anilins gossypol obtained by condensing gossypol with aniline and further
that upon hydrolysis of aniline D-gcssypl a substance was obtained which
was shown to be identical both chemically and physiologically with ana-
lytically pure gossypol.

These facts were held by Clark to render untenable the hypothesis
that D—gcssypol is an oxidation or hydrolytic product of gossypol as had
been suggested by Oarruth (93) and Sherwood (96) and point to a mechanism
such as that suggested in which gossypol is condensed with an amino group
of the protein mleculc.

Jones and Waterman (97) had previously shown that peptic and tryp-
tic digestion of casein end cottonseed globulin in vitro was reduced 15
per cent by the presence of gossypol as one per cent of the weight of
either of the proteins. They believe that incomplete digestion (83 per
cent) of cottonseed globulin by animals may be explained as due w the
inhibiting action of gossypol. This fact indirectly lends support to
the hypothesis that gossypol in cottonseed meal is bound with a group
in the protein molecule. '

Osborne and Handel (68) in 1917 had shown that rats on rations coa-
posed of cottonseed meal or cottonseed flour (41 per cent) plus starch
and lard and in nest cases butterfat had made good gains and showed no

injurious effects. Raw cottonseed kernels proved unsuccessful, the rats

dying in a very few days. However, when the raw kernels were subjected
to steam heat for two hours the toxicity was destroyed. Steaming the
raw kernels for one hour or heating in an electric oven at 110° did not
destroy the poisonous property.

Bowen and llsmul (98) autoclaved cottonseed mal at 15 pounds for
80 minutes in a damp condition and than air dried it. It was fed at
the rate of 1.33 per cent of the body weight to young pigs averaging
38.5 pounds apiece. L basal ration of dorso. skimd milk and alfalfa
was used. The pigs made fairly good gains. Pigs on a similar ration
containing unautoclaged meal showed no‘ ill effects for three weeks but
' after that they failed to grow nerneny, at vs ani 83 days, two of the
pigs on the unautoclaved weed died. It was concluded by these investi-
ptors tint autoclaving destroys the poison in cottonseed seal but whether
it is the effect of high temperature or oxidation by oxygen of the air
during drying was not discovered.

Gallup (99) autoclaved damp cold pressed cottonseed for one hour
at 20 pounds‘ pressure. He also steered some of the cottonseed ual for
one hour by passing steam directly into the meal. The treated meal was
dried and fed as 1.5 per cent of the body weight of pigs weighing approx-
imately 50 pounds. The basal ration was made up of corn chops an! al-
falfa. in the case of the pigs fed the autoclaved an! the stea-d meal
the gains were rather shall but they were considerably larger than the
gains nude by pigs in a check (lot fed untreated cold pressed seal. It
is stated, however, that the untreated meal was sparingly eaten and no
account of this apparent difference in palatability was taken in inter-

preting the results. lbcd conmtion was not inclined. Pigs receiving

tankage instead of cottonseed meal made even poorer gains than the pigs
fed on untreated meal.

Cotton seeds untreated, and cotton seeds autoclaved for one hour
and for two hours were fed to white rats as 36 per cent of the ration.
The ration was adequate from other standpoints. Autoclaving for one
hour was found to completely destroy the toxicity of tn seeds for rate.
cottonseed seal when fed to rats as 25 per cent of the ration produces
good results but 55 per cent caused injury as indicated by failure to
grow. Steaming the meal produced normal results. The addition of six
per cent butterfat, five per cent yeast, and one per cent deco: appar-
ently did nothing to overcome the toxic effect of either cotton seeds
or cottonseed meal.

The Chemistry of Gossypol

tuhlman (100) in 1861 while attempting to recover fatty acids from
the coat.» of cottonseed oil purification found in hie residue after
distillation a greenish hide use which he called ”cottonseed blue" and
gave to it the empirical formula 017112404.

Iongmrs (101) separated a very impure product from cottonseed oil
which he describes as a "light brown powder of purgent odor". It was
found to be quite insoluble in water but soluble in alcohol sill alhalies.
It was probably gossypol in a very impure state.

i ' larchlewski (102) probably first isolated gossypol in a fairly pure

'form while engaged in.the purification of cottonseed oil. He concluded

that it waspa phenol derivative and named it gossypol from Gossyp (ium

phen) ol. He proposed the formula 01331404 or 032334010. His prepare.-

tion contained loosely bound acetic acid and had a melting point of 179-
1eo° c. He described its properties as: "i beautiful crystalline yellow
colored dihydroxy phenolic substance, easily soluble in alcohol, bensene,
chloroform, ether, acetone and glacial acetic acid. Insoluble in water,
soluble in concentrated sulphuric acid with a magnificent red color,
eaaily soluble in alkalies, the solution for the first second being yel-
low and after a short time becoming a beautiful violet and then fading,
the fading being due to oxidation." Harchlewski made no crystalline
derivatives but he showed that it was not a glucoside and that in all
probability it contained no alkoxyl groups.

Withers and Garrath (87) isolated gossypol acetate from cottonseed
in 1914 but they reported, no study of its chemical properties.

Oarruth (103) in 1918 made the first extensive study of the chais-
try of gossypol. he prepared gossypol acetate by treating an ether ex.-
traction of decorticated seeds with 80 per cent acetic acid. Ihs sub-
stance was purified by dissolving the crude gossypol acetate in ether
and acetic acid arxi distilling until enough ether was driven off to al-
low the goesypol to precipitate. crystalline gossypol was obtained from
the acetate by dissolving the latter in ether and adding water. The
ether was distilled off leaving the gossypol as crusts floating on the
water which contained all of the acetic acid. The free gossypol was
then crystallized from alcohol.

Holecular weight studies were made using a method whereby the acetic
acid that was taken in) by water from an ether solution of the mssypel
acetate was titrated with stauiard alkali. From this data the amunt of

acetic acid present was obtained, and finding it to be 10.0 per cent

45

Carnth calculated the molecular weight of gossypol to be 692 if the
espirioal formula is 05032809 or 594 if its forumla is 03033009. Ecle-
cular weight determinations were made by direct titration of free' gossy-
pol with standard alkali, it being assur-d that gossypol neutralises two
equivalents of Neon. By this method the values obtained were 544 and
552. Irho fact that the disodiun salt of gossypol is neutral to phenol-
phthalein in aqueous solution gives an indication of its relative strength
as an organic acid. Analytical data give results which indicate that the
empirical formula is 63033309 or 03033009.

Complete acetylation of the gossypol was found to be difficult but
Oarruth concluded that there are four or five lurdroxyl groups in gossy-
pol. Marchlewski had supposed there were only tw0.

Aniline readily combines with gossypol to form a bright orange yel-
low compound. It was thought to be a simple dianiline compound but nit-
rogen determinations showed that it contained too Itch nitrogen to uh
this appear logical. 4.65 per cent nitrogen was found which would indi-
cate a conpound containing two molecules of gossypol to five mleoules
cf aniline. ate compound is very insoluble and therefore can be used
to determine the gossypol content of cotton seeds. Its insolubility also
probably accounts for its non toxic properties since it is probably not
appreciably absorbed in the intestinal tract.

carruth (103) found that the substance which had been hand D—gos-
sypol which is present in quite large amunts in cottonseed mal and is
insoluble in ether could be extracted froa the meal with hot aniline.
Iron the aniline extract an orange-yellow compouui crystallises out.

h-gossypol was obtained from it by dissolving the substance in alcoholic

 

 

 

\

 

potash and pouring into water, oxidation being prevented by adding a
pinch of sodium hyposulfite. D—gossypol was then precipitated in mr-
phous flakes by adding acid. The substance was then crystallised from
other and alcohol. a yellow crystalline substance was obtained which
had no definite melting point but darkened and softened around 256° 0.

Attempts to get a clue to the constitution of gossypol failed
through inability to split the substance into simpler known substances.
The fact that several flavone pignents occur in the cotton plant and the
fact that gossypol has 30 carbon atoms suggests that it may be derived
by condensation and subsequent reduction of two molecules of flavone.
The acidic properties are thought to be due to carboxyl and hydroxyl
groups arranged as in flavonals rather than to carboxyl groups. These
substances are sufficiently acid to form salts from an alkali acetate.
The presence of nine oxygen groups may readily be accounted for by the
presence of five hydroxyls, tso carboxyl groups ani two bridge oxygen
atoms, all of which types occur in flavonals.

Schwartz and. Alsborg (90) aside an Optical crystallographic study
of gossypol acetate under theflmicroscOpe. They found that in ordinary
light the substance consists of bright yellow flakes, often rather acute-
1y rhombic in outline, or sometimes approximately hexagonal. Two or
more acute crystals are sometimes grown together to form a twin with a
deep relntrant angle at one end. The crystal system is apparently tri-
clinic.- .

Clark (104) in recent work has repeated some of Oarruth's earlier
investigations on gossypol and has studied several additional derived

compounds. Gossypol was prepared by the method of Carruth. The crude

 

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47

gossypol acetate obtained was found to have a melting point of 184 -
187° c. The acetic acid was removed from the gossypol also by the method
of carruth. To purify the substance for analysis the gossypol was re-
crystallised from ether several time. The purified gossypol was fouui
to have a mlecular weight of 511. The analytical data obtained upon it
gave results which indicate the empirical formula 03035003. This differs
from the formula given by Carruth in having one less oxygen. It is pos-
sible that Carruth was working with a compouni that was not entirely pure.
The melting point of the purified gossypol was found to be 214° 0.

' When gossypol was heated to its melting point it lost two molecules
of water producing a new crystalling body. anhydrogossypol 03032505.

This is probably the same compound which Oarruth called B-gossypol.

I Oxidation of gossypol produced a colorless dioxins which seemed to
indicate that the chromofares are quinone groups.

Acetylation of gossypol showed the presence of six quroxyl groups.
to of these hydroxyl groups behave differently from the remaining four,
being Itch mre acidic and requiring drastic treatment for the hydrolysis
of their acetyl derivatives.

0f the eight carbon atoms present two are present as carbonyl groups
and tin other six as hydroxyl groups.

Olark (95) has also shown that Oarruth's aniline D-gcssypol is iden-
tical with dianiline gossypol prepared by condensation of the pure gos-
sypol with aniline, and upon hydrolysis of aniline D-gossypol, a substance
identical in its properties with pure gossypol is formed. This seems to
indicate that the structure of the gossypol mlecule is not changed in

the heating and pressing process.

Clark (105) oxidised gossypol with alkaline permanganate and ob-
tained formic, acetic and iso butyric acids as reaction products. Under
the conditions of the experiment the presence of ice butyric acid as a
decomposition product of the gossypol mlecule indicated a side chain
consisting of at least the iso butyl group.

' Clark (105) in a later intestigation treated gossypol with 4.0 per
cent sodium“ hydroxide at the temperature of the steam bath for one-half
hour. rormic acid and apogossypol, a new phenolic substance, were formed.
It has the formula (2233500, and is round by the elimination of the twe
carbowllgroups of gossypol as formic acid. The substance is a colorless
crystalline material having no definite aelting point. It ie less toxic
than gossypol when fed to white rate. It causes acute injury only, dif-
fering somewhat from gossypol in this respect.

Clark (1071 (108) had made further studies on the action of chronic
acid and aloe the'action of boiling hydriodic acid on the derivatives of
gossypol. However, no results have been obtained which elucidate the

structure of gossypol.
rormation and Occurrence of Cossypol in the Cotton Seed

Carrcth (103) refers to gossypol as occurring in interml ”resin
glands" or "oil glands" of the cottonseed.

Stanford and Viehoever (108) have nde a study of the formation and
the secretions of the internal glands of the cotton plant. These glamls
were found to be constantly present am definitely arranged throughout

the genus gossypuin.

 

 

49

In the seed the glands were found directly boneath the palisade
layerinto which they often project. 'hey are oblate-spheroidal in
fora, with long axis perpendicular to the cotyledon surfaces. anller
glands are found in the cortex of the radicle, covered by a few paren-
ehynl layers.

The glands in portions of the plant which are exposed to light are
surrounded by an anthocyan-bearing enreIOpe of the flattened cells, con-
tain quercetin probably partly or wholly in the form of its glucosides.
'i'he glands not normllyluposed to light are surrounded by a layer of
flattened cells containing no anthocyans. They contain gossypol. Gos-
sypol is formed in the glands of the denieping corolla. On their ex-
posure to light it is replaced by quercineritrin. Gossypol in the un-
folding cotyledons is changed, probably through oxidation, without the
formtion of quercimeritrin. is gossypol occurs in the glands of the
flower before Opening and quercineritrin after opening the former my
give rise to the latter. The exact nature of the change remains un-
certain. Where the glsndssre not exposed to light the gossypol ap-
parently remains unchanged.

Perkins (110) in 1909 had isolated quercimeritrin from Egyptian
cotton flowers. it was isolated. It has glistening bright yellow plates
which on hydrolysis yielded glucosennd quercetin. He concluded that one

of the following formlas indicated its structure.
I E

all a//
cit/twp 04C)” ”0 0 ‘fiQW’
ll
0%

C000” co-c-o/r

C5 //7(0h20

Viehoever (111) in 1918 showed the quercimeritrin also occurred in

' upland cotton grown'in this country.

Gallup (112) studied the gossypol content of the cotton seed dtn'ing

different stages of development. Seeds from bolls which were mature and

about to crack had an average gossypol content of .048 per cent.

Seeds

fron bolls which were Just Open had a gossypol content of .428 per cent,

after the bolls were fully opened there was a very slow increase reach-

ing a maxim of .551 per cent in mature seeds several months later.

Gallup (115) in a later study found that the develOpment of oil and

gossypol in t1: cottonseed occurred at about the same time and during a

very short and well defined period of growth, Just as the bolls were open-

ing.

with the formation of oil in the seed.

He concluded that apparently gossypol is associated in some way

Schwartsand usberg (lld) nade gossypol determinations on cotton

seeds of different varieties grown in widely separated regions.

all) made oil determinations on the same seeds.

 

 

They

The results are given

 

below.
Variety Wren Teu- ](oist- m
Grown ure Extract ~gen Pound

Lone Star Greenville, Tex. 1917 5.95 51.46 6.19 .4157 .5855 .5869
' a w fumaValley, 0.1. 1919 5.64 55.08 5.88 .4658 .4849

w 5' c-eenrine, Tex. 1919 6.65 55.97 8.50 .5507 .5884 '

.. 6v - w 1919 - 55.40 5.40 .5288 .5078

n '3 Texas 1919 5.45 54.22 5.87 .8522 .8481

s '3 Arkansas 1919. 5.84 55.56 5.84 .6849 .6770

9' w linohester, 11.0. 1919 5.68 55.45 5.82 .8592 .6899

 

1

‘W

 

 

51

 

Variety v“ EEOC Eon Tear Mist-m
Grown ure Extract gen
lone Star Courtland, Ala. 1919 5.11 58.28 5.45 .7592 .7410
7 7 7 1919 8.04 57.15 5.20 .7970 .7982
7 Bakersfield, 081. 1919 5.88 55.92 5.80 .8781 .8781
7 Elieebsth.city,n.c.1919 5.82 58.48 4.91 .9878
Durance Courtland, 118. 1919 5.88 57.18 6.27 .7178 .8970
7 Bakersfield, Cal. 1919 5.88 57.01 4.82 .8574 .8709
7 Columbia, 8. 0. 1919 5.70 58.08 5.85 .8758 .9075
7 Its. valley, Cal. 1919 5.45 57.55 5.15 .8848 .9512 .8851
7 7 7 7 1919 5.89 57.40 4.77 .9552 .9519
7 seergie,n.c.8s.c. 1919 5.58 58.87 5.19 .9811 .9849
7 Columbia, 8. c. 1918 5.95 58.97 4.94 .9845 .9855
7 Columbia, 8. c. 1917 4.81 40.80 4.881.0805 1.0548
rriee Bells, Tenn. 1918 8.12 28.87 6.42 .5970 .4250
7 7 7 1917 5.45 52.51 6.28 .5778 .5797
7 7 7 1919 8.55 55.84 5.75 .8895 .9081
7 Tennessee 1919 - - - .9428 .9524
7 Eastern States 1919 u - - 1.0590 1.1852
1881. Clarksville, Tex. 1918 5.44 55.89 8.04 .4574 .4580
7 7 7 1919 8.21 54.58 5.71 .5554 .5557
7 Oklahoma 1919 4.92 55.41 5.70 .8976 .9094 .9087
7 Bakersfield, Cal. 1919 5.25 40.98 4.40 .9859 .9612
need. Charleston, 8. c. 1918 5.52 57.05 4.97 .5858 .5741
" Ware County, Ga. 1918 5.06 57.87 5.08 .6455 .8445

 

 

P4

 

 

52

 

 

Variety Wear lioist- Wearsypol
Grown ure Extract gen Found
uliri. Eastern States 1919 5.72 58.29 5.00 1.0185
" Florence, S. C. 1919 5.74 39.53 4.66 1.0305 1.0566
Columbia Easley, s. c. 1918 5.46 58.89 4.85 1.1105 1.1182
7 South Carolina 1919 - - - 1.0064 1.0278
Egyptian Sacatone, iris. 1918 5.88 58.88 4.75 1.1852 1.1758
" Bakersfield, Cal. 1919 5.59 36.08 6.54 1.1745 1.1847
King Richmond, Va. 19197 5.24 58.55 5.14 .9219 .9185
.Cleveland Stdmthews, 8.0. 19197 6.49 55.20 5.25 .7127 .8892
Sea Island Blackshear, Ga. 1918 - - - .9446

 

From these data Schwartz and ilsberg found a positive correlation

between the ether extract content and the gossypol content (Correlation
CoefuT'equals .75 t .03. There was a negative correlation between
the nitrogen content and the ether extract. There is no relationship
between the variety and the gossypol content. The gossypol content seems
to vary considerably with the season in which it is grown and also with
the region where it is grown. The seeds from the southwest have a tem-
ency to be low in oil and gossypol, those from the southeast tend to be
higher, and those from the Pacific coast tend to be still higher.

Garner, Allard and Roubert (115) concluded that under practical con-
ditions climate is a more potent-factor than variety or soil type in con-
trolling the size of the seed and its oil content. They found as did
Schwarts and Alsberg that the oil content of the seed tended to vary with

the region in which it is grown.

 

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D —
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1
s
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s
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s
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s .v

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w
v e

 

53

Sherwood (96) analyzed 40 samples of cottonseed meal for their gos-

sypol and D—gossypol content. These data together with other information

whiCh he obtained is given in the following table.

1921 - Open Kettle

 

 

Water Time ’T'é'mp. Steels

 

 

Sam- Soil on Variety Added of of Press- mist- Cos- D-
ple Which of During CookingSeed ure in Color ure sy- Gos-
Crown cotton Cooking Min. CookerJackets % pol sy-
(°r.) lbs. 291
1 Sandy loan nixed no 24 210 - B 14.92 .045 .874
2 7 7 Cleveland yes 48 - 60 r B 6.66 .154 .755
Big Bell
3 mixed steam 12 300 90 I 6.32 .160 .669
4 Black 7 no 20 - 50 I h 7.52 .019 .919
5 Bed 7 no 25 210 - I 6.56 .021 .881
8 7 7 no 22 70 - In 8.66 .145 .985
7 (l) 84 100 I B 5.21 .020 .754
16 Saniy mixed no 7 (2) 79 100
7 (5) 100 100
7 (4) 107 100
20 (1) .
17 Clay mixed no 20 (2) 210 r B 6.04 .028 1.019
. 20 (5)
20 (4)
probably in error .
20 (1)
18 2% 20 (2) 220 r B 7.75 .009 .875
20 (5)
20 (4)
80 (1)
19 2% 20 (2) 220 I B 7.75 .009 .878
20 (5)
20 (4)

 

 

-. .4-..

 

1921 - Expeller Process

 

 

Water Tm Temp. §teani

 

 

 

Saa- Soil on Variety Added of of Press- mist- Gos- ' D-
ple Which of During CookingSeed are in Color ure sy- Gos-
Crown Cotton Cooking liin. CookerJaclnets % p01 ey-
_ KL) 1115. pol
raw kernels
20 red 8 Cleveland 8 I .58 .14
gray Big 3011
58) 182 7.18 .026 .555
59)1918 170 7.54 .020 .425
40) 160 6.59 .025 .548
Qpen Kettle Process
21 ' 40 155.250 7.75 .055 .910
22 '55 60 7.58 .051 .758
25 20 202 60 8.29 .111 .858
24 22.5 40 10.55 .078 .544
1.921 - Continuous Cooker
‘7 loan nixed no 18(1) 40 128 7.58 .054 .902
18 (2) 60
8 sandy mixed no 20 (1) 18 r 8.08 .228 .789
13 (2)
9 Coastal short 8% 58 218 80-90 I 6.45 .048 .845
plain staple
10 sandy Cooks In- no 20 (1) 175 40 r 5.74 .022 .848
loan proved . 20 (2) 176 75
20 (5) 175 90
2% 20 (1) 170 90
11 20 (2) 200 80
25 (3) 215 40 r B 6.20 .025 .811
20 (4) 220 20
12 Norfolk mixed 5% 20 (l) 180 75
lees 20 (2) 200 60 B D 5.72 .016 .808
20 (5) 220 50
20 (4) 220 30

 

a .a1 _
. .l t”
G 7 D -
s 1e) ""
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55

1921 - Continuous Cooker

 

 

 

Tater Time Temp. 5738.111
Sam- Soil on misty Added of of Press- Hoist- Cossy- D-
ple Which of During CookingSeed ure in Color are pol Gos-
Grown Cotton Cooking Min. CookerJackets sy-
(°Fg lbs. pol
20 (1) 60
13 Steam 20 (2) 60 I B 7.17 .040 .773
20 (3) 40
20 (4) 10
20 ' 5
l4 loan mixid small 20 218 10 I B 5.83 .025 .820
spray 20 to 70
20 220 65
23 (l) 220 60
15 sandy improved yes 23 (2) 220 60 I B 5.61 .328 1.014
small 23 (3) 220 60
seeds 23 (4) 220 25
25 10-12 80 9.73 .152 .584
26 20 60-80 6.97 .089 .575
27 20 8.01 .078 .932
28 20 40 7.74 .089 .754
1918 - Continuous Cooker
29 22 240 25 8.46 .037 1.076
30 60 210 5-45 8.30 .058 .832
31 24 15-35 10.12 .044 .696
32 120 226 35 12.05 .062 .984
33 72 212 15-20 8.10 .010 .751
34 . 27 50 9.05 .018 .843
35 96 25-100 10.60 .058 .633
36 27 50 8.09 .007 .999
37 75 200-230 20-60 7.21 .007 .888

 

J

 

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D I ..

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en-
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.
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.—
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7
.

 

56

THE EFFECT OF VARIOUS SUPPLEIo‘ENTS IN COUNTEBACTING THE INJURIOUS EFFECTS

OF TOXIC SUBSTANCES

Mention.has previously been.made of the work of‘Iithers and Brewster
(41), Withers and Carruth (42), and Gallup (82) which showed the value of
iron salts in counteracting or delaying the onset of the syqtoms of cot-
tonwood meal injury in.rabbit8 and swine. It was not specifically stated
by these investigators that iron has an.antidotal effect on gossypol pois-
oning, although their results point to such an interpretation.

Gallup (116) has recently published work in.whidh he shows that rats
on a diet containing 40 per cent cottonseed meal failed to grow normally
after 60 to 90 days on the ration. However, the addition of iron.in the
form of Fe 804. 7 H20 as .5 to one per cent of the ration produced normal
growth and made possible the reproduction and rearing of normal litters.
Ybry marked individual variation in susceptibility to gossypol poisoning
was noted by Gallup in this investigation.

igahur (117) in.a series of'articles (1926-1929) reported toxic ef-
tests from cod liver oils and- showed that the toxicity was greatly affected
by the composition of the basal ration.particularly its content of vita-
nin.B and C.

Slagswold (118) previously reported cases of poisoning among cattle
in Sweden which received cod liver oil. The symptoms of which.are COMP
parable to those whidh develop in.the case of cottonseed meal indury.

Light, Killer, and Prey (119) reported that the acute symptoms of
vitamin.D overdosage can be counteracted at certain levels by the liberal

feeding of yeast.

57

horrie and Church (120) in 1930 published work in which they showed
that certain cod liver oils are toxic due to their content of a poisonp
one base, iso-amylamine. They isolated this substance and fed it with
a normal cod liver oil. When the basal ration contained 10 per cent
yeast the toxic symptoms developed but when 18 per cent yeast was added
no indications of poisoning'were bbserved.

High protein diets are known to increase intestinal putrefaction
with consequent increased ptomsine formation. Hassan and Drummond (121)
in 1927 showed that the feeding of yeast and yeast extracts were bene-
ficial in counteracting the effect of an extremely high protein diet.

The beneficial effects of massive yeast feeding in counteracting
the effect of toxins in the rations suggests that perhaps the symptoms
of gossypol poisoning may be delayed or offset by preperly reinforcing
the diet, not only by the addition of iron salts but also by the use of
other supplements which may have a direct effect on the digestive appa-

ratus.

58

DISCUSSION OF REVIET.’ 0F LITERATURE

A critical analysis of the investigations pertaining to cottonseed
meal injury suggests beyond any doubt that gossypol is the toxic prinp
ciple present in cottonseed and to a lesser extent in cottonseed meal,
responsible for the injurious effect produced in swine, rabbits, guinea
pigs, and rats. The marked species difference with inspect to suscepti-
bility to gossypol poisoning is an interesting phenomenon. Swine appar-
ently are extremely susceptible to the poisoning from.gossypol and per-
haps also from D-gossypol. Rabbits and guinea pigs are also very easily
poisoned by small doses of gossypol. Bats are much more resistant to
the effects of gossypol poisoning than rabbits and swine; however, when
gossypol occurs in any appreciable amount in their diet they are poisoned.

With regard to the effects of feeding gossypol to ruminants there is
very little direct evidence to show that it is poisonous when it is fed
in the amounts in which it normally occurs in cottonseed meal. "Cottonp
seed meal injury" of cattle has been reported commonly and a belief has
grown up that cottonseed meal cannot be fed at a very high level without
producing injurious results.' However, the recent investigations at the
Nflehigan.and Nerth Carolina Agricultural Experiment Stations have shown
quite definitely that a deficiency in the ration exists when the so-called
”cottonseed meal injury” occurs and that when a ration complete in mine
drals and vitamins is used in conjunction.with cottonseed meal no in»
Jurious results are obtained.

The chemistry of gossypol is not yet clearly enough elucidated to

permit a deduction as to the structure of the gossypol molecule and it

 

 

59

is difficult therefore to explain.what kind of a reaction is responsi-
ble fer the formation of Carruth's D-gossypol in the manufacture of

cottonseed meal. It appears that Degossypol or "bound gossypol" as it
exists in cottonseed meal is much less toxic than.free gossypol as it

naturally occurs in cotton seed.

60

EXPERIMENTAL WORK
Object

Cottonseed meal furnishes a cheap source of protein for dairy cattle
rations. It has not been used extensively because it has apparently pro-
duce injurious effects with several classes of live stock.

This injurious effect has been.attributed to the presence in cottonp
seed meal of a phenolic substance called gossypol. It has been.proven
that gossypol is extremely toxic to some species of animals but its ef-
fect on dairy cattle is not well known.

The object of this experiment is to determine whether or not gossy-
pol as it occurs in raw cottonseed meal is toxic to dairy heifers where
an.adequate ration is used, and to study, with rats, the effect of adding

various supplements to a normal ration in alleviating the injury caused

by gossypol.
ORIGINAL RLAN'OF EXPERIMENT WITH DAIRY HEIFERS

Procedure
m1. Used
Five grade Holstein heifer calves will be used in this investiga-
tion. Animals G 20, G 21, and G 22 will constitute lot 1. Animal G 17

‘iill constitute lot II. Animal G 16 will constitute lot 1110

£83.
The animals will be placed on experiment at 6 months of'age and will
continue on experiment through first calving time or longer if the re-

sults up to that time indicate a need fer a continuation of the experiment.

61

Health

 

Only healthy growing heifers which have no observable abnormalities
will be used in this experiment.

Choice 2.1.: Rations

 

_l_l_i_l_l_:_ The calves of all three lots will receive whole milk until
45 days of ago. From 45 days to 180 days they will receive skim milk
in. amounts not to exceed 12 pounds per day.

_G_r_a_i_._n The heifers in let I will receive all of the raw crushed
cottonseed of known gossypol content that they will readily consume and
up to a level which will furnish all of the protein necessary for min-
tsnance and growth according to the Armsby reading Standard (122). lot
11 will receive choice cottonseed meal in sufficient quantities to furs.-
ish all the protein needed for maintenance and growth. Lot 111 will re-
ceive linseed oil reel in similar amounts. Let. I and 11 will receive
.t pounds of yellow corn per day in addition to cottonseed and cotton-
seed seal.

M Timothy hay of good quality will be fed in whatever
amunts the heifers will consume. Corn silage of good quality will be
fed with the hay. It will be offered in whatever amounts the heifers
will readily consume but it will not be fed at a level high enough so
that the amount of hay consumed is limited.

Innsrals Two per cent of special steamed bone meal and one per

cent salt will be mixed with all of the grain fed to the heifers in the

three lote.’

62

Moment

gate}; The animals in all of the lots will receive water at least
once daily until they are 15 months of age. When they are running in an
outside enclosure during the day, they will have free access to water.
After they become 15 months old they'will be placed in stanchions equipped
with drinking cups.

Shelter The calves will be housed in the College Experimental
Barn until they are 18 mnths of age. From 18 months to the time of par-
turition they will be kept in the Experimental Barn annexe In either
case the conditions of housing will be similar to those commonly found
on Michigan farms. Calves will be kept in individual box stalls until
they become large enough to be tied in individual stalls. 1t 15 months
of age they will be placed in stanchions.

_ Sunlight The animals will be exposed to direct sunlight during
the time that they are allowed to run outside). During the spring and
sumer months this will amunt to several hours each day. During the
winter months the amount of sunlight that they will receive will be very
limited due to natural conditions of cloudiness and also to the fact that
they will be confined inside most of the time. Exposure to not more than
one to two hours of sunshine per week will be the usual condition in" the
mnths of December, January, and February.

Cars The animals will be under the care of a competent feeder

 

who will have charge of the experimental barn where the heifers are

hD'IIBCd e

Peeding Methods

The animals will be fed twice daily. Silage and grain will be fed
in the morning and hay will be fed at night. The hangers will be so ar-
ranged that losses in feeding will be reduced to a minimum and feed rc-

fused will be weighed back.

COLLECTION OF DATA

Feed Records

 

M Consumed All feed fed during the experiment will be weighed
and feed refused will be weighed back. 1 daily record of all feed con-
sulnd will be kept. This will be totaled for 30 day periods and placed
in table form.

Nutrients Required {me nutrients required for maintenance and
growth will be calculated for each 50 day period from the ersby Feeding
Standard. The weight to be used in figuring the nutrients required will
be obtained from the mnthly weight records.

nutrients Consumed The nutrients consumed will be calculated
from the average daily consumption of each 30 day period. These will be
arranged in table form.

Per cent .93 Raw Cottonseed Meats _i_n_ the Ration The per cent of

 

 

raw cottonseed meats in the ration will be computed on the basis of the
total food intake by 50 day periods. All feeds will be converted to a
dry basis using the Henry and Morrison (123) tables for misture content.

Amount 23 Cosmol Ingested The amount of gossypol consmned per

 

day will be calculated using the per cent of gossypol found in samples

of the raw cottonseed meats fed.

64

Per cent of Cosszpol in the Ration The per cent of gossypol in

 

the ration will be calculated for each 30 day period for the heifers in
lot I.

My: Cosszpol maggwm The milligrams of
gossypol ingested per kilo of body weight per day will be calculated from
the data obtained,using the weight for the end of each period as the ba-
sis for computing the kiIOgrams of body weight. for each of the animals
in lot I.

Reproduction

 

Oestrml Periods The date of the appearance of the first heat
period and each subsequent heat period will be recorded.

Breeding 251?. The date or dates upon which each animal is bred
will be recorded. A competent veterinarian will examine each animal to
determine whether conception has occurred. m abnormalities in the.
reproductive organs as found by the veterinarian will be noted.

Ease 35 Parturition Notes will be made at the time of calving

 

on the ease with which the calf is delivered.

Placenta A record will be made of the time after parturition
at which the cow delivers the placenta. In case the placenta is re-
tained mre than 24 hours it will be removed by a competent veterinar-
ian. In case of an abortion a part of the placenta will be plated by
a bacteriologiet to determine whether or not a causative organism is
present.

Recovery The condition of the animal after parturition will be
carefully watched for several days. Any abnormlity in health or con-

dition will be recorded.

65

Offspring The weight, strength, and general physical condition
of the offspring will be recorded. at the time of parturition. The eyes
of the calf will be tested to determine whether or not the vision is im-

paired.

Observations

 

m The animals in all the lots will be weighed at 10 day
periods until they are 15 months of age. After that time they will be
weighed at 30 day intervals. The weights used for computing nutrients
required will be taken from an average of three weighings taken on con-
secutive mnrings of each 30 day period. The animals will be weighed
in the warning before they are fed. Increase in weights will be shown
by graphs compared to Eckles normal.

fight at Withers The height at withers will be determined every
30 days. in average of three measurements will be taken with the animl
being moved between each determination. The increase in height at with-
ers will be shown in graphical form compared to the Eckles normal.

Consistency 93 Feces The consistency of feces will be determined

 

for a 14 day period on each animal by the method develcped by Moore and
Huffm’n.

Health Any abnormalities in the health of the animals will be

 

investigated by a veterinarian and the results of his findings will be
recorded.
Qpetite The appetite of the animals during the experiment will

be watched by the feeder in charge. He will record any indication of

loss of appetite.

66

Blindness The animals will be watched very carefully for any
indication of blindness, and the condition if found will be recorded

in detail.

 

Stiffness and. Abnormalities 3.32311 The heifers will be ob-
served by the feeder in charge while they are in the outside yard. Any
condition of stiffness or irregularity in gait will be recorded.

Page}; CarryingOCapacity of the M The oxygen carrying capac-
ity of the blood will be determined on each animal every month after the
heifers are 15 maths old. The gasometric method of Van Slyke (124)
will be used. These analyses will be made by the Department of Experi-
ment Station Chemistry. .

Photographs Photographs will be made of the heifers at six months,

15 mnths, and at the time of parturition.

PROCEDURE

Animals 9393

The animals were used as originally planned in this experiment.
£2.

The heifers in let I were started on experiment at eight months of
age instead of at six months as was originally planned.

_R_e_a_l_1_;_l_i_ The heifers were all in good health at the start of the
experiment.
m 25 Rations

Milk ' Milk was fed as originally planned.

 

Grain Grain was fed as originally planned. Raw cottonseed con-
taining an average of .8 per cent "free gossypol" was obtained for the

experiment and was used throughout.

 

.. lenlll Jill]. .IIIII:

67

Roughage Roughage was fed as planned. The quality of the silage
fed during the latter part of July and the first part of August, 1950,
was rather poor. The quality of silage was reduced at that time due to
a shortage in supply.

Minerals Iinerals were supplied as originally planned.
mnegentent

Water Water was supplied as originally planned, except that from

 

October of 1950 until the completion of the trial, when the animls were
housed in the experimental barn annex, which is not equipped with a water-
ing system, the water was supplied in an outside tank.

Shelter The shelter used was as originally planned.

Beddig Wood shavings and sawdust were used throughout the trial
for bedding.

Exercise Exercise was allowed as planned.

Sunlight The animals were allowed the benefit of whatever sunlight
there was during the time whenweather conditions permitted their being
out doors.

Care Care was given as originally planned.

 

leeding lethod The feeding method originally planned was followed

throughout the experiment.

DATA.

Peed Record
Feed Consumed The record of feed consumed by 30 day periods by

all animals is included in Tables I and V.

68

Nutrients Required The nutrients required as computed from the
Armby Feeding Standard is included with the tables of feed consumed.

Nutrients Consumed The amount of nutrients consumed is included
with the above data in Tables I to V. Digestible crude protein and net
energ values from Armsby's tables were used in computations of nutri-
ents consumed except in the case of raw cottonseed meats. In the case
of the raw cottonseed the digestible crude protein value was obtained
by multiplying the nitrogen found on analysis by 6.25 and taking 70 per
cent of the protein as digestible, (Henry and Harrison (123M). 1 value
of 17.16 per cent crude digestible protein was thus obtained. The net
' energy value was assumed to be approximtely 115 therms, considering the
raw cottonseed meats to contain 56 per cent oil which was found to be the
case on analysis.

Per cent 33 Raw Cottonseed Meats in the Ration The per cent of

 

 

raw cottonseed meats in the ration computed on the amount of dry material
in the ration for each 30 day period after the animals in let I were
placed on trial is shown in Tables V to VIII.

Mg; Gosszpol Ingested The amount of gossypol ingested with
the raw cottonseed meats bythe animals in let I is included in Tables
7 to VIII. Computations of the amunt of gossypol ingested is on the
basis of the average per centage of gossypol found in samples of the raw
cottonseed meats used. This amounted to .8 per cent.

Per cent 3_f_ Gossypol in the Ration The per cent of gossypol in

 

the ration of the heifers in lot I is included with the above data.

69

Nilligrams 23 Gossflol per Kilo 9_f_ 1303 Weight The milligrams
of gossypol per kilo of body weight per day is included with the above
data.

Reproduct ion

 

Oestrua} Periods The occurrence of the first oestrml period is
recorded and each consequent period is noted in Table II for all of the
animals in all lots.

Breeding 931:: The dates on which the heifers were bred is in-
cluded in Table IX. None of the heifers in either of the three lots
have freshened at the time that thee data were assembled, hence the ob-
servations covering parturition cannot be included.

Observations

 

Eight The weight of the individual animals in each lot, taken
by 30 day periods compared to the Eckles normal, is shown in graphs I to
V. The weight by 50 days periods is also shown in tabular form in Table
I.

2.15—1.13. 31 Withers The growth in height at withers of all the
animls in the three lots canpared to the Eckles normal is shown in
graphs I to V. The height at withers taken by 30 days periods is also
included in Table 1.

Consistency _o_f_ Feces The consistency of feces of the animls in

 

the three lots over a period of 14 days is shown in Table 11.

Health The health of the taninals in all of the three lots was

good throughout the emeriment. There was no apparent difference in
health of the animals in lot I as compared to the two check animls. They

have maintained a good condition of flesh since the start of the experi-

70

ment, and it was impossible to find any evidence of malnutrition in their
general appearance.

gpetite The appetite of the two check animls in let II and lot
III was good throughout the experiment except for one instance when G 1?
went off feed for a period of a few days. Its appetite returned to nonnl
without treatment, however, and it has continued to eat normally.

The heifers in lot I have had a fairly good appetite during the em- I
periment but have on a few occasions gone off feed. It was found impos-
sible to feed as high a level of raw cottonseed meats as was originally
planned, because the unpalatable nature of the meats caused the animals
to eat them sparingly on several occasions.

Blindness There was no indication of impaired vision in my of
the animals used in this investigation.

Stiffness _an_d_ Abnormalities in. 9331 The animls in let I have
shown no stiffness in their Joints, no swelling in their legs, nor any
noticeable abnornnlities in gait at any time during the experiment.

953.32 Laying Capacity 95 the; M Table III shoes the oxygen
carrying capacity of the blood as indicated by its hemoglobin content
for all of the animals on experiment.

Photographs The photographs of the animals taken at the start
of the experiment and at 15 months of age are included in plates I to

VIII.

71

MERILEHTS WITH RATS

While a very marked species difference is known to exist in sus-
ceptibility to gossypol poisoning, as has been previously pointed out
it was thought desirable to study the effects of gossypol poisoning on
rats, using raw cottonseed meats from the same lot of material that was
used. in feeding the dairy heifers. The toxicity of the raw cottonseed
meats could thus be compared to a known value, for raw cottonseed kernels
as well as gossypol have been fed to rats by several investigators.

It is also possible in working with rats to study the antidotal ef-
fect of various supplements to a basal ration which contains a sufficient
amount of gossypol to be injurious to the health of the animals. The
amunt of time and expense connected with such an investigation in large
animals practically prohibits the use of dairy cattle.

The object of the feeding trials with rats was to check the toxicity
of the cottonseed meats used in the ration of the dairy heifers by feed-
ing the material directly and by isolating the gossypol from it and feed-
ing it in an otherwise normal diet. It was further desired to study the
effect of various supplements to the basal diet used in protecting the

animals against poisoning from gossypol.

Animls Used
The rate used in this series of trials were albino and piebald ani-
nls. No animals which were known to be cross bred were used in any of

the trials.

72

g The animals were started on eXperiment when they were approx-
imately 28 days of age. Up to the time that they were 28 days of age
they had been left with the mother and were allowed to eat the same sim-
ple ration which she received.

Weight The animls averaged from 50 to 60 grams in weight when
started on the trials except in the case of series I in which heavier
animals were used. In each series the lots were so arranged that the
average weight of each lot was nearly the same as the average for all
of the lots.

£95 Both male and female animals were used in these trials. When-
ever growth weights were compared animals of the same sex were used. All
graphs showing increase or decrease in weight will be labeled with the
sex of the animal.
lbnagement

£s_g_esUsed Wire cages about 10 inches in diameter and 12 inches

 

high with screen bottoms which allowed the feces and urine to pass through
were used in all of the trials.

m if. Feeding Feed was kept before the animals at all tines.
No attempt was made to measure accurately feed consumption but the appe-
tite of the animals was observed by the relative amount of food eaten
daily and by the tendency of some aninals to waste feed when the appetite
became impaired.

Water Distilled water was kept before the animals at all times.

Diets Used

 

 

Series I This series was run to check the toxicity of the ration

 

73

which was being fed to the dairy heifers on the trial previously reported.
The possibility that alfalfa hay carried a factor or factors which might
alleviate the injurious effects of gossypol was studied. Raw cottonseed
meats taken from the same lot of feed as that fed to the dairy heifers

was used as 25 per cent and 50 per cent of the diet. The diets used were:

G 4 G 5
raw cottonseed meats 25 raw cottonseed meats 25
alfalfa meal 20 yellow corn 75
yellow corn 55 steamed bone meal 1
steamed bone meal 1 salt 1
salt 1
G 6 G 7
raw cottonseed meats 25 raw cottonseed meats 25
rolled 08.158 73 alfalfa 20
steamed bone meal 1 rolled oats 53
salt 1 steamed bone meal 1
salt 1
G 8
linseed oil meal 25
alfalfa meal - 2O
rolled oats 55
steamed bone meal 1

salt

F1

 

74

Series E}. This series was run to determine the effect of feeding
cod liver oil and large quantities of yeast with rations containing .2
per cent and 0.1 per cent gossypol acetate. The basal ration used was
assumed to be adequate for good growth.and the supplements added were in
excess of those demanded for an adequate diet which contained no toxic
substances.

G W 1. Positive control - basal diet
yellow corn. 44

whole wheat flour 40

alfalfa meal 4
casein 10
Ca 003 1
salt 1

c‘w 2. Negative control
Basal diet plus .2 per cent gossypol acetate
G‘W 3. Negative control
Basal diet plus .1 per cent gossypol acetate
GVW 4.
Basal diet plus .2 per cent gossypol acetate plus two per cent
cod liver oil.
G W'S.
Basal diet plus .1 per cent gossypol acetate.plus two per cent
cod liver oil.
GVW.5.
Basal diet plus .2 per cent gossypol acetate, 15 per cent whole

wheat replaced by 15 per cent yeast.

75

GWW 7.

Basal diet plus .1 per cent gossypol acetate, 15 per cent whole

wheat replaced by 15 per cent yeast.

Series £££_ This series was a study of the effect of yeast, cod
liver oil and butterfat when added to an.adequate diet in.a11eviating
the toxicity of gossypol. Raw cottonseed meats containing approximately
.8 per cent gossypol were used at various levels.

G C 1. Positive control - basal diet

rolled oats 84
casein 10
alfalfa meal 4‘
Ga 00$ 1
Na01 1

G G 2. Negative control
rolled oats V 74

raw cottonseed 10

casein 10

alfalfa meal 4

Ca 003 1

Heel 1
G G 3.

Diet G c 2 plus 2 per cent cod liver oil
G C 4.
Diet‘G O 2.with 10 per cent rolled oats replaced by 10 per cent

powdered yeast.

G G 5. Negative control
rolled cats 69

raw cottonseed meats 15

casein 10
alfalfa meal 4
Ca. 003 1

seal 1
G c 6.

Diet G O 5 plus two per cent cod liver oil

G 0 7.
Diet G 0 5 with 10 per cent rolled oats replaced by 10 per cent
powdered yeast

G G 8.
Diet G 0 5 with 15 per cent rolled oats replaced by 15 per cent
powdered yeast

G C 9.
Diet G G 5 with 10 per cent rolled oats replaced by 10 per cent
butterfat -

G C 10. Negative Control
rolled cats 64

raw cottonseed meats 20

casehn 10
alfalfa meal 4
0‘ 003 1

N301 1

 

H

 

77

G c 11.
Diet G C 10 with 10 per cent rolled oats replaced by 10 per cent
yeast.

G O 12.
Diet G C 10 with 15 per cent rolled oats replaced by 15 per cent
yeast.

G C 13.
Diet G O 10 plus two per cent cod liver oil. After one week on.this
diet the animals were changed to a diet in.which 15 per cent rolled
oats was replaced by 15 per cent yeast. Two per cent cod liver oil
was added as before.

G O 14. Negative control
rolled cats 59

raw cottoneeed meats25

casein. lO

alfalfa meal 4

Ca 003 1

N301 l
G G 15.

Diet G C 14*with 10 per cent rolled oats replaced by 10 per cent
yeast.
G 0 16.

Diet G c 16 with 15 per cent rolled oats replaced by 15 per cent

yeast.

78

Collection of Data

[sight The animals were weighed individually when they were
placed on emeriment and were weighed every seven days thereafter as
long as the trial was continued.

Growth The amount of growth measured by increase in weight from
week to week is shown in graphical form in graphs VI to XXXII.

M The growth curve has been used entirely as a measurement
of gossypol injury, although there have been other indications of poison-
ing. Loss of appetite has been one of the first indications of injury.
it high levels of intake the injury was acute and death resulted before
other symptoms set in. Where chronic injury develcped, there was usually
extreme irritability. An emaciated condition developed accompanied by a

loss of hair on the neck am shoulders.

Determination of Gossypol in Samples of Cottonseed Neal and
Raw Crushed Cottonseed Meats

Collection 93 Samples Five southern oil companies located at various

 

places in the southern states collected samples from the regular mill
run of the 1929 crop. Twelve samples of cottonseed meal and 12 samples
of raw crushed cottonseed meats, ready for the cooker were collected in
the Spring of 1950. The samples of meal and the sample of meats are not
in all cases from corresponding "batches”. The analysis of the samples
was made in the summer of 1950.

liethod _o_f $191: The samples of raw crushed cottonseed meats and

 

cottonseed meal were ground finely enough so that the material would

79

pass through a 10 mesh sieve. The gossypol content was determined by
the aniline method of Carruth (103).

Fifty grams of the material was extracted.with ethyl ether in.a
continuous extractor, of the Soxhlet type, until the distillate was per-
fectly colorless. The ether was then.driven off and-the oil which re-
mained was diluted with three volumes of petroleum.ether and thoroughly
mixed. This solution.was allowed to stand in.cylinders until the ins
soluble resinous materials settled out. ‘A 75 per cent aliquot of the
liquid was removed from.the cylinder and placed in.a.small precipitating
bottle. Three cubic centimeters of aniline were added and the material
was set aside for two weeks to allow complete precipitation to take place.
The precipitated Dianiline gossypol was recovered on.a gooch crucible,
washed thoroughly with petroleum ether and dried at 1000 c. The weight
of the Dianiline derivative multiplied by .775 was taken.as the amount of
gossypol present in the aliquot of the oil. The amount of gossypol pres-
ent in the cottonseed meal and in the raw crushed cottonseed meats was
expressed in terms of per cent by weight.

Results The data recorded are shown.in Table XIII.

Because of the fact that the cottonseed meal samples analyzed were
not from the same "batch” as the raw cottonseed meats no attempt has been
made to correlate gossypol content in the finished meal with gossypol con-
tent in the original seeds. However, a close observation.of these data
indicates that there is probably a relationship between the gossypol conp
tent of the finished meal and the conditions under which it was processed.

The mill using cottonseed meats having the lowest gossypol content of any

 

luljllu.l

80

analysed, furnished samples of meal having a higher gossypol content
than the average of meals from all of the mills.

It will be observed that none of the samples of cottonseed meal
analysed had a gossypol content of more than five one hundredths of one
per cent and there are but eight samples having a gossypol content over
for one hundreths of one per cent. This consistently low gossypol con-
tent in samples of meal from various regions and different mills indi-
cates that there is almost no possibility of getting a lot of cotton-
seed meal that will contain more than six per cent of the amount that
has been present in the raw crushed cottonseed meats which have been fed

without injury to growing dairy heifers.

Discussion of Experimental Results

A study of Tables I to V showing feed consumed, nutrients required
and nutrients consumed shows that the animls in lot I, G 20, G 21, and
G 22, received less digestible protein than the amount required accord-
ing to the Armsby Staniard. During several thirty day periods the de-
ficiency in amount of protein as compared with the requirement was con-
siderable. This deficiency in amount of. protein was due largely to the
fact that the heifers would not eat a sufficient amount of raw cotton-
seed meats to furnish the necessary protein. The raw cottonseed meats
were unpalatable having a rather bitter residual taste and being very
high in oil. The energy furnished by the ration fed to the animals of
lot I was above the requirements during most of the thirty day periods
included in the experiments. The rations of the heifers in lot 11 and
lot 111 furnished an excess of protein and a small excess of energy

throughout the experiment.

 

H

{A

 

81

Daspite the fact that the heifers in lot I received less digestible
protein.than those in.Lot II and lot III their growth was very comparable.
All of the heifers were below the Eckles normal in weight and height at
withers during a part of the growing period. .it two years of age, hose
ever, all of the animals were normal or above, as compared with the Eckles
standard, and there was no significant difference between the heifers of
lot I and the two check animals in.growth.

The ration.of the heifers in let I contained on the average from.20
to 25 per cent raw cottonseed meats during the experiment as Tables VI,
III, and VIII indicate. This amount of raw cottonseed meats represented
approximately .2 per cent of gossypol in.the ration. The amount of’gossyb
pol in the ration was three times the amount feund to be toxic to rats
by Schwartz and.Alsberg (90) and four times that found to be toxic to
rats by Clarke, yet the heifers showed no indication.of injury. In.visw
of the fact that rats were found relatively much less susceptible to gos-
sypol poisoning than rabbits and swine, this level of two per cent of
gossypol in.the ration.of growing heifers indicates a very high species
tolerance for gossypol.

I On the basis of milligrams of gossypol per kilo of body weight conp
sumd daily the heifers in lot I.had a lower intake of gossypol than was
feund to be the threshhold of toxicity for rate by Schwartz and ilsberg.
The average for all of the heifers in lot I throughout the experiment was
31.8 milligrams of gossypol per kilogram.of body weight, which is about

one-half the amunt found to be toxic to rats by the above cited experi-

mentors.

82

There was no significant differences between the heifers in lot I
and the two check animals in the oxygen.carrying capacity of the blood
as indicated by its hemoglobin content.

In the experiments with.rats series I, a diet similar to the ration
fed the heifers was used in which a 25 per cent level of raw cottonseed
meats was used. As graphs VI to II show, the rats lost weight steadily
and died in from 10 to 28 days or were killed because of their extremely
emaciated condition. .A diet high in.vitamin.i (6’4 and G 7) showed no
superiority over one low in.vitamin A (G 6) in:lessening the injurious
effects produced by the cottonseed meats. Vitamin A'was furnished'by
alfalfa hay which was used as 20 per cent of the diets of G 4 and G 7.

The failure of the heifers to show any injury in response to high
gossypol feeding may'be accounted for on two premises. First, that in
the digestive tract of the ruminant conditions exist such that gossypol
is not absorbed, or if it is absorbed, is changed chemically so as to be
less toxic, or second, that the gossypol intake per unit of body weight
was not high enough in.this experiment to reach the threshhold of toxic-
ity. In.view of the fact that swine, rabbits, and guinea pigs are pois-
oned by very small quantities of gossypol per unit of body weight and be-
cause there are not clear out cases of cottonseed meal injury in ruminants
which can‘be directly traced to gossypol poisoning it seems quite likely
that the failure of the heifers to show any injury from.gossypol feeding
is due to conditions along the digestive tract, peculiar to the ruminant.

What these conditions may be cannot readily be suggested.

83

The results of gossypol analysis on samples of cottonseed meal which
show a consistently low content of gossypol in the meal. from various mills
indicates that there is practically no possibility of the dairyman getting
a meal which contains enough gossypol to be toxic to dairy cattle even
though it be fed in excessively large amounts.

Thrmghout the experiment the feces of the heifers in lot I were mch
harder in consistency than those of the two check animals. Whether or not
this difference is due to the presence of gossypol in the ration is not
known but it seems likely that the condition causing the hard consistency
may be associated with the presence of a large amount of cottonseed oil
which with its predominance of fatty acids of high molecular weight may
upon saponification form heavy soapy mterial that tends to make hard well
formed feces.

This condition which arises from the feeding of raw crushed cotton-
seed meats having a high oil content may in part explain why cottonseed
meal has in the past been considered costive although its effect as has
been shown by Huffman and Moore is actually laxative.

The results with rats, series 11 and series III, indicate clearly
that where gossypol is fed at a comparatively low level but at a level
which st ill causes injury, the addition of yeast as 15 per cent of the
diet decreases the injurious effect to such an extent that the animls
grow normally. God liver oil used as two per cent of the diet relieved

the inJury to a lesser extent but the beneficial effect did not appear

to be permanent.

The effect of yeast cannot be attributed to bolstering a vitamin B

84

deficient diet. The diet used in series II and series III was probably
not deficient in vitamin B. The effect of yeast on the intestinal tract
is far reaching. It is well known that it changes the intestinal flora
and in the review of literature it was pointed out that it is beneficial
in counteracting the 111me occurring from the feeding of high protein
diets which cause intestinal putrefaction with consequent formation of
ptomaines. It was also shown that it is effective in counteracting the
toxic effect of iso amlamine in the diet. The mechanism of its bene-
ficial effect has not been explained in these cases nor can it be ex.-
plained in this connection but it seems likely that its effect is re-
stricted to conditions along the intestinal tract.

The temporary beneficial effect of cod liver oil in relieving the

injurious effect produced by gossypol feeding cannot readily be explained.

85

Summary

1. Raw crushed cottonseed meats (gossypol content eight tenths per
cent) were fed in amounts varying from 20 to 25 per cent of a ration
containing silage and good quality hay without producing injury in grow-
ing dairy heifers. No lameness, swollen legs or impaired vision re-

sulted. The hemoglobin content of the blood was not decreased.

2. Gossypol analysis of cottonseed meal samples showed that a. maxi-
mum of .05 per cent gossypol may normally occur. This is but six per
cent of the amount present in the raw crushed cottonseed meats used

in the experiment.

3. The feeding of raw cottonseed meats to dairy heifers resulted in
the formation of hard well formed feces, an effect distinctly differ-

ent from that produced by the feeding of cottonseed meal.

f.

4. Rate died in 10 to 28 days when fed a diet containing the same pro-
portions of raw cottonseed meats that were fed without injury to heifers.

Equivalent amounts of crude gossypol acetate produce similar results.

5. The addition of nassive doses of yeast (15 per cent) to a diet con—-
taining an injurious amount of gossypol, alleviated the injurious effect

and allowed for practically norml growth in rats.

1.

2.

3.

4.

5.

6.

7.

8.

86

LI TERIEURE CITED

Royal Society of Arts, London.

1783 Transactions of Society Instituted at London for Encourage-
ment of Arts, Manufactures and Commerce with the Premiums
Offered. 1783 Vol. I, p. 251, cited by khcy.

Jour. of Dairy Sci. Vol. 4, p. 250-257.

Bishop, J. 1..

1785 A History of American manufactures from 1608 to 1860.
Vol. 2, p. 81. Edward Young a 00., Philadelphia.

United States Yearbook of Agriculture, 1930.

1950 U. 8. Government Printing Office. p. 65

Voelker, A.

1872 Jour. Royal Society of England. Vol. 8, pt. I, p. 219, cited
by Kilgore. U. s.'Expt. Sta. Bul. 35, p. 421. ‘

Stone, W. E.

1889 Cottonseed Hulls and Esal as Food for Live Stock.

Tenn. Agr. Expt. Sta. 3111. Vol. 2, No. 5.

Wright.

1889 Arkansas Agr. Expt. Sta. Bul. No. 9.

Lloyd, E. R.

1890 Feeding for Milk and Butter
Mississippi Agr. Expt. Sta. Bul. No. 13, p. 8.

Chamberlain, J. R.

1891 Cottonseed Bulls and Meal for Steers

North Carolina Sta. Bul. 81, pp.3.

a.

$‘—1

‘7

 

9.

10.

11.

12.

13.

14.

15.

87

Tietze
1893 Poisoning Calves by Feeding Cottonseed Meal
liilch. Ztg. Vol. 23, No. 3, pp. 38459. 1894.
Abs. In Expt. Sta. Rec. Vol. 5, p. 826.
Enery, F. E.
1894 Feeding Trials with Lnimals
. North Carolina Agr. Exp. Sta. Bul. 109, p. 419
Gautier, D. and C. F. Larsen.
1895 landmandsbade Vol. 28, p. 128-150.
Abst. in Expt. Sta. Rec. Vol. 7, p. 252.
Emery, E. E. and B. W. Kilgore
1895 Cottonseed Hulls and Meal for Beef Production
North Carolina Aer. Expt. Sta. Bul. 11s.
Connell, J. H. and H. C. Kyle
1899 Feeding Steers - Feed Value of Cottonseed and its Products
Texas Agr. Exp. Sta. Bul. 55, p. 131-214.
Rust
1905 Poisoning by Cottonseed Meal
Wrofferitl. Johres. Vet. Ber. Lauraitse Preuss 6, 1905, pt. 2
pp. 35-36, publ. 1908.
Abs. in Expt. Sta. Rec. Vol. 21, p. 788-790.
Michele, J. and J. l. Burgess
1906 1 Comparison of Wheat Bran arxi Cottonseed Meal for Hill: Produc-
tion.

8. Carolina Agr. Exp. Sta. Bul. Vol. 117, pp. 18.

 

.11-].1

16.

17.

18.

19'.

20.

21.

88

McNutt, J. C.
1911 Report of the Division of Dairy Husbandry
No. Carolina 361'. Exp. Sta. Annual Rpt. Vol. 34, p. 33.
Dinwiddie, R. R. and L. K. Short
1911 Cottonseed lieal Poisoning of Livestock
Arkansas Agr. Exp. Sta. Bul. 108.
Moore, J. S.
1914 Feeding Cottonseed Meal and Hulls to Dairy Cows
mssissippi 131‘. Exp. Sta. Bul. 174
Halverson, J. 0. and F. W. Sherwood
1950 Investigations in the Feeding of Cottonseed Heal to Cattle
N. Carolina Agr. Expt. Sta. Tech. Bul. 39
Reed, 0. 3., C. F. Huffman and L. H. Addington
1927 Cottonseed Meal as a Feed for Dairy Calves
Jour. Dairy Sci. Vol. 11, Ho. 6, pp. 488-515

Huffman, C. F. and L. L. more

, 1930 Cottonseed Meal Studies - Heavy Feeding of Cottonseed lieal

to Dairy Cattle hiring Reproduction and Lactation
Jour. of Dairy Sci. Vol. 13, Ho. 6, pp. 478-494
Ruffmn, c. s. end 1.. i. more
1929 Effect of Cottonseed Meal and Linseed Oil Meal on the Con-
sistency of Feces of Dairy Cattle
Jour. Dairy Sci. Vol. 12, No. 5, pp. 410-418
Von Nathausius
1886 Fuhlings lands Ztg Vol. 35, p. 114, cited by Kilgore

Us Se EXPte Sta. 3111. Vol. 53, p. 420

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29.

30.

31.

89

Esser

1894 Landw. Were. Stat. Vol. 42, p. 303, cited by Kilgore
U. s. Expt. Ste. 3111. as

Ladd, E. F.

1888 Report of the Chemist

-89 New York Agr. Expt. Sta. Annual Rpt. 1888-9, p. 300

Jones, J. l. and R. F. Dickson

1928 Grain Sorghums versus Corn for Fattening Lambs
Texas Agr. Expt. Sta. Bulletin 379

Gray, D. T. and J. W. Ridgway

1909 Feeding Cottonseed Meal to Pregnant Ewes
Alabam Expt. Sta. Bul. 148

Cox, R. F.

1929 Cottonseed Meal with Various Roughages as Fattening Rations
for Lambs.
New Mexico Agr. Expt. Sta. Bul. 179

Burkett, C. W.

1903 Feeding Farm Horses and Mules.
N. Carolina Agr. Expt. Sta. Bul. 189

Templeton, G. S.

1927 Mule Feeding Experiment
Mississippi Agr. Expt. Sta. Bul. 244

3611, C. L. and J. 0. Williams

1920 Cottonseed Meal for Horses

U. Se Dept. Of Agr. Bill. 929

32.

53.

34.

55.

35.

37.

58.

39.

Curtis, G. W. and J. W. Carson

1892 Effects of Cotton Seed and Cottonseed meal in.Feeding Hogs.
Texas Agr. Expt. Sta. Bul. 21

Carrcevin, C.

1896 L Poison in cottonseed and Cottonseed Cake.
Ann. Agron. ve1. 22, no. 8, p. 353-561
Abs. in.Expt. Sta. Rec. vo1. 8, p. 155

Dinwiddie, R. R.

1903 Pig Feeding Experiments with Cottonseed Meal
Arkansas Agr. Expt. Sta. Bul. 76

Georgeson, C. 0., F. C. Burtis and D. H. Otis

1895 Feeding Experiments with Pigs on Corn, Wheat, Kafir and
Cottonseed Meal
Kansas Agr. Expt. Sta. Bill. 53

Curtis, C. F.

1895 Feeding Cottonseed Meal and Other Heals to Hogs
Iowa Agr. Expt. Sta. Bul. 28

Duggar, J. F.

1903 Grazing and Feeding Ezq>erirnents with Pigs
‘Llabama Agr. Expt. Sta. Bul. 122.

marshall, F. R.

1905 Feeding Fermented Cottonseed Meal to Hogs
Texas Agr. Expt. Sta. Bul. 78

Curtis, R. S.

1909 Feeding Fermented Cottonseed.Mea1 to Hogs

N. Carolina Agr. Expt. Sta. Bul. 200.

41.

42.

45.

91

Walker, J. B.

1916 Some Experiments and Practical Demonstrations in Hog Feed-
ing at the Delta Branch Experiment Station
Miss. Agr. Exp. Sta. Bill. 177.

Withers, W. F. and. J. F. Brewster

1913 Studies on Cottonseed Meal Toxicity. II. Iron.as an Antidote
Jour. Biol. Chemistry, Vol. 15, No. 1, p. 161.

Withers,'w. F. and F. E. Carruth

1917 Iron as an.Antidote to Cottonseed Meal Injury
Jour. Biol. Chemistry, vol. 32, no. 2, p. 245.

Rommel, G. M. and E. B. Vedder I

1915 Beri beri and Cottonseed Meal Poisoning in Pigs
Jour. of Agr. Research, Vol. 5, p. 489.

Wells, C. A. and P. V. Ewing

1916 Cottonseed Meal as an Incomplete Food
Jour. Biol. Chem. Vol. 27, p. 15.

Goldberg, S. A. and L. A. Maynard

1922 Studies in Cottonseed Meal Poisonings I. The Pathological
Tissue Changes Resulting from Continuous Feeding of Cotton-
seed Meal
Am. vet. Med. Ass'n Jour. Vel. 62, p. 450.

Barnett, E. and C. J. Goodell

1923 Grazing and Feeding Trials with Hogs

Mississippi Agr. Exp. Sta. Bul. 218, p. 17.

 

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47.

48.

49.

50.

51.

52.

55.

92

Warren, G. R. and Williams, D. 27.
1923 Swine Feeding Experiments. III. Cottonseed and Cottonseed
Meal for Fattening Pigs
Texas Agr. Exp. Sta. Bul. 305, p. 25-27.
Bethke, R. 11., G. Bohstedt, H. L. Sassaman, D. C. Kennard and B. H.
Edington
1927 The Comparative Nutritive Values of the Proteins of Linseed
Heal and. Cottonseed Meal for Different Animals
Jour. of Agr. Research, Vol. 36, p. 853.
Hale, Ffed
1930 Cottonseed Meal as a Feed for Hogs
Texas Agr. Exp. Sta. Bul. 40.
Souls, A. 11. and J. R. Fain
1905 Stall Feeding versus Grazing
Virginia Agr. Exp. Sta. Bul. 164, p. 51-88.
Roberts, 1. P. and J. E. Rice
1897 Cottonseed Meal, Shorts and Wheat Bran for Chickens
New York (Cornell) Exp. Sta. Bul. 87.
Thompson, R. B.
1918 Poultry Feeding Experiment
New Mexico Agr. Exp. Sta. Bul. 117.
Sherwood, R. M.
1928 Effects of Various Rations on the Storage Quality of Eggs

Texas Agr. Exp. Sta. Bul. 376.

 

 

54:.

55.

56.

57.

58.

59.

50.

93

Symes, ‘J. L. and J. A. Gardner

1915 Toxicity of Sodium PyraphOSphate Administered in Food
Biochemical Journal, Vol. 9, p. 9-16.

McCallum, E. 7., M. Simmonds and W. Pitz

1916 The Distribution in Plants of the Fat Soluble A: The Dietary
Essential of Butterfat
Am. Jour. of Physiology, Vol. 41, p. 361.

Richardson, Anna E. and Helen S. Green

1916 Nutritional Investigations upon Cottonseed Meal 1.
Jour. Biol. Chemistry, Vol. 25, No. 2, p. 307-318.

Richardson, Anna E. and Helen S. Green

1917 Nutritional Investigations upon cottonseed l-‘Eeal II.
Jour. Biol. Chem. Vol. 50, No. 2, p. 245.

Osborne, T. B. and L. B. Mendel

1917 The Use of Cottonseed as a Food
Jour. of Biol. Chem. Vol. 29, p. 289-317.

Nevins, W. B. .

1921 The Proteins of Cottonseed Meal, II. Nutritive Value
Jour. Dairy Sci. Vol. 4, p. 552-588.

Gallup, 'w. n.

1928 The Digestibility of the Proteins of Some Cottonseed Meal
Products

Jour. Biol. Chem. Vol. 76, p. 43-53.

61.

62.

63.

65.

66.

67.

68.

94

Bdhm, R.

1881 Address before the Scientific Society of mrburg
U. S. Exp. Sta. Bul. 33, p. 96.

Ritthausen and ’."ieger

1882 J. Pragt Chem. Vol. 30, p. 32
U. S. Expt. Sta. Bul. 33, p. 96.

Cachtgens

1870 Dar pater med. Zeitschrift Ed. I cited by TI. Mamell
Am. Chem. Jour. Vol. 13, p. 469.

liaxwell, W.

1891 On the Nitrogenous Bases Present in the Cottonseed
Am. Chem. Jour. Vol. 13, p. 469.

Von Hoeslin

1906 Beit Zur. Chem. Physiol. u. Pathol, Vol. 8, p. 27
Cited by Macy - Jour. Dairy Sci. Vol. 4, p. 250-257.

Frieman

1909 Inaugural Dissertation, University of Bern.
U. S. Exp. Sta. Bul. 33.

Edgerton, C. "J. and H. Morris

1912 Some Studies of Cottonseed Meal Poisoning.

Kanig Spieckermann and Olig

1903 Zeitschrift far Untursuschung der Lrahrungs Genussmittel VI,
No. 5, p. 193, No. 6, p. 241, HO. 7, p. 289

Cited by Racy - Jour. Dairy 3C1. V01. IV, N0. 3, P0 2500

69.

70.

71.

72.

73.

74.

75.

76.

95

Hardin, M. B.

1892 On the Available Phosphoric Acid and the Water-Soluble Potash
in Cottonseed Meal
South Carolina Agr. EXP. Sta. Bul. 8.

Crawford, A. C.

1910 Editorial

Exp. Sta. Record, Vol. 12, p. 503.

 

Anderson '5'" ‘I
1912 The Organic PhOSphoric Acid of Cottonseed Meal
Jour. Biol. Chemistry, Vol. 13, p. 311.
Withers, W. A. and B. J. Ray
1915 Studies in Cottonseed Meal Intoxication, I. PerphOSphoric Acid
Jour. Biol. Chem. Vol. 14, p. 53.
Forbes, E. B. and F. M. Beagle
1915 Organic and Mineral Analysis of Foods
Ohio Agr. Exp. Sta. Bul. 255, p.226.
Wells, C. A. and P. V. Ewing
1915 Acidosis and Cottonseed Heal Injury
Georgia Agr. Exp. Sta. Bul. 119, p. 35-64.
Withers, 17. A. and F. E. Carruth

1917 Gossypol the Toxic Substance in Cottonseed

 

Jour. Agr. Research, Vol. 12, p. 83-101.
Bechdel, S. I., C. H. Eckles, and L. S. Palmer
1926 The Vitamin B Requirements of the Calf

Jour. Dairy Sci. Vol. 9, p. 409-438.

! Wanwmmims-r gist}; 'i - ' _'

 

(\

4"

77.

78.

79.

80.

81.

82.

83.

84.

96

Bechdel, S. 1., H. E. Honeywell, R. infiltcher and. M. H. Knutsen
1928 Synthesis of Vitamin B in the Rumen of the Cow
Jour. Biol. Chem. Vol. 80, No. l, p. wl-238.
Easy, 1. G. and Ia. B. Mendel
1930 Jour. Pharm. and Exp. Therap., Vol. 16, p. 345.
Richardson, A. s. and H. s. Green
1917 Nutrition Investigations upon Cottonseed Meal
Jour. Biol. Chem. Vol. 31, p. 379-399.
Stevens, Henry
1930 Nutrition Studies on Cottonseed Meal
Progress report presented before the meeting of American Oil
Chemists Society, New Orleans, Kay 9, 1930.
McGowan, J. P. and Chricton
1924 Cottonseed Meal Poisoning
Biochemical Jour. Vol. 18, p. 273—282.

Gall-tip, g. D.
1928 The Value of Iron Salts in Counteracting the Toxic Effects

of Gossypol
Jour. Biol. Chem. Vol. 77, p. 437.

Bachdel, s. 1., s. E. Honeywell and a. A. Dutcher

1928 The Effect of Feeding Heifers a Ration Deficient in Vitamin A
Pennsylvania Sta. Bul. 230, p. 20-23.

Jones, I. R., C. H. Eckles, and L. S. Palmer

1926 The Role of Vitamin A in the Nutrition of Calves

Jour. Dairy Sci. Vol. 9, No. 2, p. 119-139.

 

85.

86.

87.

88.

89.

90.

91.

92.

97

Steenbock, H. and Gross
1919 Jour. Biol. Chemistry, Vol. 40, p. 501.
Withers,‘W. A. and B. J. Ray
1912 A lethod for the Removal of the Toxic PrOperties from Cotton-

seed.Meal. A.preliminary report. Science, vol. 36, No.914.
Withers, W.A. and F. E. Carmth
1914 Gossypol a Toxic Substance in Cottonseed

A preliminary note, Science, Vol. 41, No. 1052, p. 524-1915.
Withers, W. A.
1915 Gossypol the Toxic Substance in Cottonseed Nbal

Jour. Agr. Research vol. 5, p. 261-288.
Schwarta, E. W. and C. L. Alsberg
1924 Pharmacology of Gossypol

Jour. Agr. Research, 761. 28, p. 191-198.
Schwartz, E.‘W. and C. L. Alsberg
1924 Relation.between Toxicity of Cottonseed and its Gossypol

Content

Jour. Agr. Res. 761. 28, p. 173-189.
menaul,rP.
1923 The Physiological Effects of Gossypol

Jour. of Agr. Research, vol. 26, p. 233.
Withers, av. A. and r. E. Carruth
1918 Comparative Tbxicity of Cottonseed Products

Jour. Agr. Research, Vol. 14, p. 425-452.

 

93.

94.

95.

96.

97.

98.

99.

Withers, W. A. and F. E. Carruth

1918

Clark,

1928

Clark,

1928

Gossypol, the Toxic substance in Cottonseed

Jour. Agr. Researdh, Vol. 12, p. 83.

E. P.

The Composition.and Toxic Effects of Gossypol

Oil and Fat Industries, Aug. a Sept., 1928, p. 237-277.
E.IP.

Studies on Gossypol. II. Concerning the Nature of Carruths
D-Gos sypol

Jour. Biological Chemistry, 761. 76, Nb. 1, p. 229.

Sherwood, F.‘3.

1926

Jones,

1923

Dowell,

1923

The Gossypol and D—Cossypol Content of Some NOrth Carolina
Cottonseed Esals

Jour. Agr. Research, Vol. 52, p. 795.

D. B. and H. C. watermen

Studies on the Digestibility of Proteins in.Vitro. If. 0n
the Digestibility of Cottonseed Globulin and the Effect of
Gossypol upon the Peptic and Tryptic Digestion.of Proteins.
Jour. Biol. Chem. 761. 56, p. 501-511.

C. T. and P. manaul
Effect of Auto claving upon the Toxicity of Cottonseed Meal

Jour. Agr. Research, 761. 26, p. 11.

Gallup, We Do

1926

Eliminating the Toxicity of Cottonseed Meal

Jour. Dairy Science, 761. 9, p. 359-372. ‘

 

100.

101.

102.

103.

104.

105.

106.

99

Kuhlman, F.

1861 Compt. Bend. 761. 53, p. 444-1861, cited by Carruth
Jour. Am. Chemical Society, Vol. 40, No. 1, p. 647.

Longmore, J.

1886 Cottonseed Oil, Its Coloring matter and mncilage
Jour. Soc. of Chem. Industries, 761. 5, p. 200-206.

marchlewski, L. P. T.

1899 Gossypol ein Bestandtheil der Baumwollsamen
Jour. Prakt. Chem. 1;. F. Bd. 60, Heft. g; p. 84-90.

Carruth, F. E.

1918 Contribution to the Chemistry of Gossypol - The Toxic
Principle of Cottonseed
Jour. Am. Chem. Society, Vbl. 40, p. 647-663.

Clark, E. P.

1927 Studies on Gossypol. I. The Preparation, Purification, and
Some of the Pr0perties of Gossypol, the Toxic Principle of
Cottonseed.

Jour. of Biological Chemistry, vol. 75, No. 3, pp. 725.

Clark, E. P.

1928 Studies on Gossypol. III. The Oxidation of Gossypol
Jour. Biological Chemistry, Vol. 77, NO. 1, p. 81.

Clark, E. P.

1928 Studies on Gossypol, IV. Apogossypol

Jour. Biol. Chem. vol. 78, No. 1, p. 159-166.

 

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107.

108 .

109 .

110.

111.

112.

100

Clark, E. P.

1929 Studies on Gossypol. V. The Action of Chronic Acid upon
Some Gossypol Derivatives
Jour. Am. Chem. Soc. Vol. 51, p. 1475.

Clark, E. P.

1929 Studies on Gossypol. VI. The Action of Boiling Hydriodic
Acid as Used in the Zeisel Method upon Gossypol and Some
of Its Derivatives. A Semi micro Zeisel Hethoxyl Method
Jour. Am. Chem. Soc. Vol. 51, p. 1479.

Stanford, E. E. and A. Viehoever

1918 Chemistry and Histology of the Glands of the Cotton Plant,
with notes on the Occurrence of Similar Glards in other
Plants
Jour. Agr. Res. Vol. 13, p. 419-436.

Perkins, A. G.

1909 Gossypium Herbaceum. Part II. The Coloring Matter of
Cotton Flowers
Jour. Chem. Soc. London, 701. 95, pt. 2, p. 2181-2193.

Viehoever, A., L. H. Chernoff and C. 0. Johns

1918 Chemistry of the Cotton Plant with Special Reference to
Upland Cotton

Jour. Agr. Research, Vol. 13, p. 345.

Gallup, .W. D.

1927 The Gossypol Content and Chemical Composition of Cottonseeds
During Certain Periods of Development
Jour. Agr. Research, Vol. 34.

113.

114.

115.

116.

117.

118.

101

Gallup, W. D.

1928 A Chemical Study of the Development of Cotton.Bolls and
the Rate of Formation of Gossypol in Cottonseed
Jour. of Agr. Research, V61. 36, PP. 471.

Schwartze, E. W. and C. L. Alsberg

1923 Quantitative variation of Gossypol and its Relation to
the 011 Content of Cottonseed
Jour. Agr. Research, V61. 25, p. 285-295.

Garner, W. W., H. A. Allard and C. L. Foubert

1914 Oil Content of Seeds as Affected by nutrition of the Plant
Jour. Agr. Research, V61. 3, p. 227.

Gallup, s. D.

1931 Concerning the Use of Cottonseed.Nbal in the Diet of the Rat
J6ur. Biol. Chem. V61. 91, p. 387.

Agduhr, E.
Acta Paediat. Vol. 5, p. 319 (1926), V61. 6, p. 165 (1926),
vol. 7, p. 299 (1929), vol. 8, p. 364 (1929), Vol. 9, p. 170
(1929), Cited by Norris and Church.
Jour. Biol. Chem. V61. 89, p. 437 (1930).

Slagswold, L.

1925 Cod Liver 011 Poisoning in Calves
Earsk Veterinaer Tidsskrift, V61. 6, p. 161-168.

Abst. J. Am. Vet. med. Assn. V61. 21, p. 236.

119.

120.

121.

122.

123.

124:.

Light, 3. F., G. Miller and C. N. Frey

1929 Studies on the Effect of Overdosage of Vitamin D.
Jour. Biol. Chem. Vol. 84, P. 487.

Norris, E. R. and A. E. Church

1930 Toxic Effect of Fish Liver Oils
Jour. Biol. Chem. p. 437-448.

Hassan, A. and J'. C.Drummoni

1927 The Physiologin Role of Vitamin B. Part IV.
Biochemical Jour. V01. 21, p. 653.

Armsby, H. P.

102

1917 The Nutrition of Farm Animals, lst Edition, p. 713, table b.

rl‘he Macmillan Company, New York.
(a) Henry, W. A. and F. B. Morrison

Feeds and Feeding, 19th Edition, p. 709-721

rJIhe Henry and Morrison Company, Madison, Wisconsin.

(1:) p. 722.
Van Slyke, D. D. and “if. C. Stadie
1921 The Determination of the Cases of the Blood

Jour. Biol. Chem. Vol. 49, p. 1-42.

103

APPENDIX

104

 

 

 

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115

Table x1. Consistency of Feces of the Heifers in all Lots for a

Fourteen Dag'Period

 

 

 

 

Lot 111 Lot 11 Lot I
3516 M 20 c: 2r fie
Date Linseed Cottonseed Raw Cotton. Raw Crushed Raw Crushed
011 Meal Meal seed Cottonseed Cottonseed
march 29 .65 .80 .55 .50 .25
50 .85 .50 .50 .25 .20
51 .60 .55 .55 .25 . .25
April 1 .90 .80 .30 .50 .15
2 .55 .55 .50 .25 .15
5 .70 .80 .40 .25 .20
4 .70 .70 .55 .25 .40
5 .40 .50 .45 .55 .20
6 .80 .70 .65 .50 .50
7 1.10 .70 .40 .50 .50
8 .80 .65 .40 .55 .20
9 .70 .55 .55 .50 .20
10 .65 .40 .50 .25 .20
11 1.00 .65 .45 .40 .55
Ave. for
14 days .745 .652 .596 .295 .259
Aye. for
Lot 1 .509
Standard
1.5 very soft .75 average
1.25 medium soft .50 medium.hard
1.00 soft .25 very hard

.15 exceedingly hard

 

 

 

116

Table XII. Hemoglobin Content of the Blood of All the

Animals for Last Nine months on.Experiment

 

 

 

Animl Animal Animal Animal Animal

G 20 G 21 G 22 G 17 G 16
hemoglobin Imglobin hemoglobin hemoglob in bemoglob in

#per cent ‘per cent <per cent per cent Aper cent

8- 6-50 77.8 82.9 79.2 75.6 80.7 t
I 9- 8-50 74.4 71.5 65.8 74.6 82.?
10-15-30 55.0 79.7 73.5 '73.3 81.0
11-19-50 86.4 80.2 81.2 91.0 85.?
12-11-50 84.6 90.0 88.7 89.7 101.1
1-14-51 86.5 86.5 99.0 90.7 96.5
2-11-51 89.1 87.0 85.0 88.5 94.7
5-11—51 89.0 77.8 81.9 95.5 75.0
4-24-51 86.9 92.6 92.6 96.2 90.9

 

Ill!"

117

Table XIII. Per cent of Gossypol in.Samp1es of Cottonseed

Meal an). Ear Crushed Cottonseed Meats

 

Sam- 'f’er cent of 38am- Per cent of Gossypol
Region from.whioh. ple Gossypol tn ple in.RaI Crushed Cotton,

 

'§=ggles tore Obtained N0. 0. 8. ML Nb. seedlnsats _4_
north Caroline 1 .0244 1 .621
" ' 2 .0159 2 .560
" ' 5 .0255 5 .815
” " 4 .0155 4 .681
" " 5 .0159 5 .684
" " 6 .0025 6 .592
7 " 7 .0225 7 .840
" ” 8 .0195 8 .664
" " 9 .0154 9 .764
” ” 10 .0218 10 .871
7 8 11 .0259 11 .859
' ” 12 .0250 12 .661
South Carolina 15 .0415 15 .807
” " 14 .0575 14 .778
” " 15 .0295 15 .716
" " 16 .0202 16 .749
" " 17 .0191 17 .856
” ” 18 .0526 18 .768
" 7 19 .0295 19 .782
" ” 20 20 .779
” ” 21 .0259 21 .696
" '.' 22 .0288 22 .847
" " 25 .0425 25 .804
" ” 24 .0455 24 .761
Mflssissipp 25 .0501 25 .657
" " 26 .0254 26 .607
' ” 27 .0544 27 .765
" " 28 28 .711
” ” 29 .0521 29 .676
" ” 50 .0581 50 .642
" " 51 .0295 51 .725
" ” 52 .0202 52 .645
" " 55 Trace 55 .592
" " 54 .0592 54 .575
2 " 55 .0414 55 .750
” ” 56 .0245 56 .658
Gnahoma 57 .0158 57 .585
" 58 .0169 58 .497
9 59 .0162 59 .500
" 40 .0250 40- .541
" 41 .0248 41 .656
' 42 42 .585

 

 

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.1

.1

Table XIII.

(Continued)

 

.Region.fronxih1dh

EEEE}" were Obtained Nb. C. S. M5 N0. seed usats

Oklahoma 45 .0096 45 .620
” 44 Trace 44 .602
" 45 .0211 45 .568
” 46 .0204 46
7 47 .0265 47 .542
" 48 48 .612

Tex: 49 .0447 49 .545
' 50 .0499 50 .265
7 51 .0472 51 .557
” 52 52 .542
" 55 .0568 55 .277
' 54 .0516 54 .280
" 55 .0579 55 .504
” 56 .0428 56 .251
” 57 .0555 57 .245
" 58 .0596 58 .246
" 59‘ .0544 59 .501
” 60 Trace 60 .242

Sam- Fe—r cent of 5m— Per cent of Gossypol
ple Gossypol in. ple

118

 

in Raw Crushed Cotton-

 

 

MICHIGAN STATE COLLEGE

119

 

 

\ MICHIGAN STATE COLLEGE

44-0

8 bargmmangor MAQEMATICD

 

eptmod

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00
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\ MICHIGAN STATE COLLEGE

 

\.

MICHIGAN STATE COLLEGE

 

MICHIGAN STATE COLLEGE

 

\ MICHIGAN STATE COLLEGE

 

,_ MICHIGAN STATE COLLEGE 125

 

\ MICHIGAN STATE COLLEGE

 

DEPARTM ENT OF MATHEMATICS

 

 

MICHIGAN STATE COLLEGE

l I'Ilj

DEPARTM ENT OF MATHEMATICS

128

MICHIGAN STATE COLLEGE

\

 

MICHIGAN STATE COLLEGE 129

 

 

\ MICHIGAN STATE COLLEGE

 

\ MICHIGAN STATE COLLEGE 3

 

 

 

 

 

 

 

 

 

 

 

 

 

   

 

 

   
       
     
      
   
       
   

 

 

 

   
 

  
 

 

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8 8 g 8 g m DEl-{EZRTMEN13F MATBEMATICS

 

MICHIGAN STATE COLLEGE

 

133

. Plate I.

40 60 80 I00 IZO I40 I68 I8_O 7 200 ZZQ

 

Animal G 20 at Start of Experiment
Ration - Raw Cottonseed Meats,
Timothy Hay, Silage and Corn.

Plate II.

20 4o 60 30 I00 :20 mo I60 I80, 200 220 240 260 280 3c

 

I80

 

 

 

 

 

 

Animal c: 20 at 15 Months of Age.

1254

Plate III.

20 40 60 Ev' :00 :PO IAO 160 180 200 229

 

 

 

 

 

 

 

 

Animl G 21 at Start of kperimnnt
Ration - Bu Cottonseed Meats,
Timthy Hay, Silos. and Corn

Plato IV.

20 4O 60 80 IOO IZO I40 I60 ISO 200 22C} 240

 

 

 

 

 

 

 

Aninl G 21 at 15 lbntha of Leo

Plot. V.

 

Lain]. G 22 at Start of Experiment
notion - Bu cottonseed floats,
rimth; my, Silage and. Corn.

Plato VI.

 

Animal G 22 ot 15 lbntha of Age

‘ ._‘1
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135

136

Plats VII.

20 40 so so IOO '20 I40 169_ I}

   

Animal G 16 at Three Months of Age

Plate VIII.

 

Animal G 16 at 15 Months of Age.
notion - Linseed Oil Iss1, Timthy
Hay and Silage

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